Methods for producing an immune response to tuberculosis

ABSTRACT

Methods for producing an immune response to  Mycobacterium tuberculosis  (Mtb) are disclosed herein. In several examples, the immune response is a protective immune response. In additional embodiments, methods are disclosed for inhibiting an infection with Mtb, preventing an infection with Mtb, or treating an infection with Mtb. Pharmaceutical compositions for the inhibition, prevention and/or treatment of tuberculosis are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 14/590,810,filed on Jan. 6, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/510,862, filed on May 18, 2012, now issued asU.S. Pat. No. 8,961,989, which is the §371 U.S. National Stage ofInternational Application No. PCT/US2010/057479, filed Nov. 19, 2010,which was published in English under PCT Article 21(2), which in turnclaims the benefit of U.S. Provisional Application No. 61/263,206, filedNov. 20, 2009. The prior applications are incorporated herein byreference in their entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No.HSSN266200400081C awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD

This application relates to the field of immunology, more specificallyto methods for the production of an immune response to tuberculosisantigens in a subject.

BACKGROUND

Mycobacterium is a genus of aerobic intracellular bacterial organismsthat, upon infection of a host, survive within endosomal compartments ofmonocytes and macrophages. Human mycobacterial diseases includetuberculosis (caused by M. tuberculosis), leprosy (caused by M. leprae),Bairnsdale ulcers (caused by M. ulcerans), and various infections causedby M. marinum, M. kansasii, M. scrofulaceum, M. szulgai, M. xenopi, M.fortuitum, M. chelonae, M. haemophilum and M. intracellulare (seeWolinsky, Chapter 37 in Microbiology: Including Immunology and MolecularGenetics, 3rd Ed., Harper & Row, Philadelphia, 1980).

One third of the world's population harbors M. tuberculosis and is atrisk for developing tuberculosis (TB). In immunocompromised patients,tuberculosis is increasing at a nearly logarithmic rate, and multidrugresistant strains are appearing. In addition, mycobacterial strainswhich were previously considered to be nonpathogenic strains (e.g., M.avium) have now become major killers of immunosuppressed AIDS patients.Moreover, current mycobacterial vaccines are either inadequate (such asthe BCG vaccine for M. tuberculosis) or unavailable (such as for M.leprae) (Kaufmann, Microbiol. Sci. 4:324-328, 1987; U.S. Congress,Office of Technology Assessment, The Continuing Challenge ofTuberculosis, pp. 62-67, OTA-H-574, U.S. Government Printing Office,Washington, D.C., 1993).

Inhibiting the spread of tuberculosis requires effective vaccination andaccurate, early diagnosis of the disease. Currently, vaccination withlive bacteria is the most efficient method for inducing protectiveimmunity. The most common mycobacterium employed for this purpose isBacillus Calmette-Guerin (BCG), an avirulent strain of Mycobacteriumbovis. However, the safety and efficacy of BCG is a source ofcontroversy and some countries, such as the United States, do notvaccinate the general public.

Mycobacterium tuberculosis (Mtb)-specific CD4⁺ and CD8⁺ T cells arecritical for the effective control of Mtb infection. In the mouse model,passive transfer of CD4⁺ T cells to sublethally irradiated animalsrenders them less susceptible to Mtb infection (Orme, J. Immunol.140:3589-3593, 1988). Mice in which the gene(s) for CD4 or for MHC ClassII molecules are disrupted, as well as wild-type mice depleted of CD4⁺ Tcells, demonstrate increased susceptibility to Mtb infection (Flory etal., J. Leukoc. Biol. 51:225-229, 1992). In humans, humanimmunodeficiency virus-infected individuals are exquisitely susceptibleto developing TB after exposure to Mtb, supporting an essential role forCD4⁺ T cells (Hirsch et al., J. Infect. Dis. 180:2069-2073, 1999). CD8⁺T cells are also important for effective T cell immunity (see Lazarevicand Flynn, Am. J. Respir. Crit. Care Med. 166:1116-1121, 2002). Inhumans, Mtb-specific CD8⁺ T cells have been identified in Mtb-infectedindividuals and include CD8⁺ T cells that are both classically HLA-Iarestricted (see, for example, Lewinsohn et al., J. Immunol. 165:925-930,2000) and nonclassically restricted by the HLA-Ib molecule HLA-E(Lewinsohn et al., J. Exp. Med. 187:1633-1640, 1998). However, there areno vaccines or therapeutic strategies that effectively induce an immuneresponse, such as a CD8 response, to Mtb.

SUMMARY

Accordingly, there is a need in the art for agents that can produce animmune response to Mtb that can be used for treatment and/or protectionfrom an Mtb infection.

Methods for producing an immune response to Mycobacterium tuberculosis(Mtb) are disclosed herein. Methods for treating an Mtb infection,inhibiting an Mtb infection, or preventing an Mtb infection in asubject, are also disclosed herein. The Mtb infection can be latent oractive.

In several embodiments, the methods include administering to the subjecta therapeutically effective amount of a polypeptide, or a polynucleotideencoding the polypeptide, wherein the polypeptide comprises at least oneof the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, orSEQ ID NO: 18. In additional embodiments, the methods includeadministering to the subject a therapeutically effective amount of apolypeptide comprising at least nine to twenty consecutive amino acidsof at least one of the amino acid sequences set forth as SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, or SEQ ID NO: 18, wherein the nine to twentyconsecutive amino acids specifically bind major histocompatibilitycomplex (MHC) class I. In some examples, the polypeptide includes aconservative variant of the polypeptide (for example, one or moreconservative amino acid substitutions). In several examples, the immuneresponse is a protective immune response. In additional embodiments,methods are disclosed for inhibiting or preventing an infection withMtb, or treating an infection with Mtb.

Isolated polypeptides are described herein that include nine to twentyconsecutive amino acids of at least one of the amino acid sequences setforth as SEQ ID NOs: 1-18, wherein the nine to twenty consecutive aminoacids specifically bind major histocompatibility complex (MHC) class I,wherein the isolated polypeptide does not include any of the full lengthamino acid sequences set forth as SEQ ID NOs: 1-18. Nucleic acidsencoding these polypeptides, vectors including these nucleic acids, hostcells including these nucleic acids, and immunogenic compositionsincluding these polypeptides, nucleic acids and/or host cells are alsodisclosed. Pharmaceutical compositions including a therapeuticallyeffective amount of these polypeptides, nucleic acids, and/or host cellsare also described.

The foregoing and other features will become more apparent from thefollowing detailed description, which proceeds with reference to theaccompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a pair of bar graphs showing percent positive samples in theELISPOT assay for the indicated antigens in individuals with latent oractive TB.

FIG. 1B is a pair of graphs showing spot forming units (SFU) for eachantigen in the ELISPOT assay. The antigens are listed in Table 2.

FIG. 2A is a bar graph showing percent positive samples for fiveselected antigens by CD8 ELISPOT assay in individuals with latent oractive TB.

FIG. 2B is a graph showing SFU/250,000 CD4/CD56 depleted PBMC by ELISPOTassay. For each antigen, “L” indicates individuals with latent TBinfection and “A” indicates individuals with active TB infection.

FIG. 3 is a graph showing SFU for each antigen in the ELISPOT assay.

FIG. 4 is a graph showing SFU for peptides covering amino acids 137-151of Rv3136.

SEQUENCE LISTING

The nucleic acid and amino acid sequences listed herein or in theaccompanying sequence listing are shown using standard letterabbreviations for nucleotide bases, and one letter code for amino acids.Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand.

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named 899-81443-15_Sequence_Listing.txt, Oct. 17, 2016, and is117 KB, which is incorporated by reference herein.

SEQ ID NOs: 1-18 are the amino acid sequences of Mtb polypeptides.

SEQ ID NOs: 19-36 are the nucleic acid sequences of polynucleotidesencoding the Mtb polypeptides.

DETAILED DESCRIPTION

Methods for producing an immune response to Mycobacterium tuberculosis(Mtb) are disclosed herein. In several examples, the immune response isa protective immune response. In additional embodiments, methods aredisclosed for inhibiting an infection with Mtb, or treating an infectionwith Mtb. Pharmaceutical compositions for the inhibition, preventionand/or treatment of tuberculosis are also disclosed.

I. ABBREVIATIONS

APC: antigen presenting cell

BCG: Bacillus Calmette-Guerin

DC: dendritic cell

HLA: human leukocyte antigen

IFN-γ: interferon-γ

MHC: major histocompatibility complex

Mtb: Mycobacterium tuberculosis

TB: tuberculosis

II. TERMS

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include asuspension of minerals (alum, aluminum hydroxide, or phosphate) on whichantigen is adsorbed; or water-in-oil emulsion in which antigen solutionis emulsified in mineral oil (Freund incomplete adjuvant), sometimeswith the inclusion of killed mycobacteria (Freund's complete adjuvant)to further enhance antigenicity (inhibits degradation of antigen and/orcauses influx of macrophages) Immunostimulatory oligonucleotides (suchas those including a CpG motif) can also be used as adjuvants (forexample see U.S. Pat. No. 6,194,388; U.S. Pat. No. 6,207,646; U.S. Pat.No. 6,214,806; U.S. Pat. No. 6,218,371; U.S. Pat. No. 6,239,116; U.S.Pat. No. 6,339,068; U.S. Pat. No. 6,406,705; and U.S. Pat. No.6,429,199). Adjuvants include biological molecules (a “biologicaladjuvant”), such as costimulatory molecules. Exemplary adjuvants includeIL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2,OX-40L and 4-1 BBL

Amplification: Use of a technique that increases the number of copies ofa nucleic acid molecule (e.g., a DNA or RNA molecule) in a specimen. Anexample of amplification is the polymerase chain reaction, in which abiological sample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of amplification canbe characterized by electrophoresis, restriction endonuclease cleavagepatterns, oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen,such as an Mtb polypeptide.

A naturally occurring antibody (e.g., IgG, IgM, IgD) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. However, it has been shown that theantigen-binding function of an antibody can be performed by fragments ofa naturally occurring antibody. Thus, these antigen-binding fragmentsare also intended to be designated by the term “antibody.” Specific,non-limiting examples of binding fragments encompassed within the termantibody include (i) a Fab fragment consisting of the VL, VH, CL and CH1domains; (ii) an F_(d) fragment consisting of the VH and CH1 domains;(iii) an Fv fragment consisting of the V_(L) and V_(H) domains of asingle arm of an antibody, (iv) a dAb fragment (Ward et al., Nature341:544-546, 1989) which consists of a VH domain; (v) an isolatedcomplementarity determining region (CDR); and (vi) a F(ab)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region.

Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture (e.g., see U.S. Pat. Nos.4,745,055 and 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP125,023; Falkner et al., Nature 298:286, 1982; Morrison, J. Immunol.123:793, 1979; Morrison et al., Ann. Rev. Immunol. 2:239, 1984).

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. In one embodiment, T cells respond to the epitope, when theepitope is presented in conjunction with an MHC molecule. Epitopes canbe formed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5, about 9, or about 8-10 aminoacids in a unique spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance.

An antigen can be a tissue-specific antigen, or a disease-specificantigen. These terms are not exclusive, as a tissue-specific antigen canalso be a disease-specific antigen. A tissue-specific antigen isexpressed in a limited number of tissues, such as a single tissue. Atissue-specific antigen may be expressed by more than one tissue, suchas, but not limited to, an antigen that is expressed in more than onereproductive tissue, such as in both prostate and uterine tissue. Adisease-specific antigen is expressed coincidentally with a diseaseprocess. Specific non-limiting examples of a disease-specific antigenare an antigen whose expression correlates with, or is predictive of,tuberculosis. A disease-specific antigen can be an antigen recognized byT cells or B cells.

Antigen presenting cell (APC): A cell that can present an antigen to Tcell, such that the T cells are activated. Dendritic cells (DCs) are theprinciple APCs involved in primary immune responses. Their majorfunction is to obtain antigen in tissues, migrate to lymphoid organs andpresent the antigen in order to activate T cells.

When an appropriate maturational cue is received, DCs are signaled toundergo rapid morphological and physiological changes that facilitatethe initiation and development of immune responses. Among these are theup-regulation of molecules involved in antigen presentation; productionof pro-inflammatory cytokines, including IL-12, key to the generation ofTh1 responses; and secretion of chemokines that help to drivedifferentiation, expansion, and migration of surrounding naive Th cells.Collectively, these up-regulated molecules facilitate the ability of DCsto coordinate the activation and effector function of other surroundinglymphocytes that ultimately provide protection for the host.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

CD4: Cluster of differentiation factor 4, a T cell surface protein thatmediates interaction with the MHC Class II molecule. CD4 also serves asthe primary receptor site for HIV on T cells during HIV infection. Cellsthat express CD4 are often helper T cells.

CD8: Cluster of differentiation factor 8, a T cell surface protein thatmediates interaction with the MHC Class I molecule. Cells that expressCD8 are often cytotoxic T cells. “CD8⁺ T cell mediated immunity” is animmune response implemented by presentation of antigens to CD8⁺ T cells.

Conservative variants: A substitution of an amino acid residue foranother amino acid residue having similar biochemical properties.“Conservative” amino acid substitutions are those substitutions that donot substantially affect or decrease an activity or antigenicity of theMycobacterium polypeptide. A peptide can include one or more amino acidsubstitutions, for example 1-10 conservative substitutions, 2-5conservative substitutions, 4-9 conservative substitutions, such as 1,2, 5 or 10 conservative substitutions. Specific, non-limiting examplesof a conservative substitution include the following examples (Table 1).

TABLE 1 Exemplary conservative amino acid substitutions OriginalConservative Amino Acid Substitutions Ala Ser Arg Lys Asn Gln, His AspGlu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val LysArg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr TyrTrp; Phe Val Ile; Leu

The term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide, or that an immune response can begenerated against the substituted polypeptide that is similar to theimmune response against the unsubstituted polypeptide, such as aMycobacterium antigen. Thus, in one embodiment, non-conservativesubstitutions are those that reduce an activity or antigenicity.

Consists Essentially Of/Consists Of: With regard to a polypeptide, apolypeptide consists essentially of a specified amino acid sequence ifit does not include any additional amino acid residues. However, thepolypeptide can include additional non-peptide components, such aslabels (for example, fluorescent, radioactive, or solid particlelabels), sugars or lipids. A polypeptide that consists of a specifiedamino acid sequence does not include any additional amino acid residues,nor does it include additional non-peptide components, such as lipids,sugars or labels.

Contacting: The process of incubating one agent in the presence ofanother. Thus, when a cell is contacted with an agent, the cell isincubated with the agent for a sufficient period of time for the agentand the cell to interact.

Costimulatory molecule: Although engagement of the T cell receptor withpeptide-MHC delivers one signal to the T cell, this signal alone can beinsufficient to activate the T cell. Costimulatory molecules aremolecules that, when bound to their ligand, deliver a second signalrequired for the T cell to become activated. The most well-knowncostimulatory molecule on the T cell is CD28, which binds to either B7-1(also called CD80) or B7-2 (also known as CD86). An additionalcostimulatory molecule is B7-3. Accessory molecules that also provide asecond signal for the activation of T cells include intracellularadhesion molecule (ICAM-1 and ICAM-2), leukocyte function associatedantigen (LFA-1, LFA-2 and LFA-3). Integrins and tumor necrosis factor(TNF) superfamily members can also serve as costimulatory molecules.

Cytokine: Proteins made by cells that affect the behavior of othercells, such as lymphocytes. In one embodiment, a cytokine is achemokine, a molecule that affects cellular trafficking. Specific,non-limiting examples of cytokines include the interleukins (IL-2, IL-4,IL-6, IL-10, IL-21, etc.), and interferon (IFN)-γ.

Degenerate variant: A polynucleotide encoding an epitope of an Mtbpolypeptide that includes a sequence that is degenerate as a result ofthe genetic code. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are included in this disclosure as long as the amino acidsequence of the Mtb polypeptide encoded by the nucleotide sequence isunchanged.

Dendritic cell (DC): Dendritic cells are the principle APCs involved inprimary immune responses. DCs include plasmacytoid dendritic cells andmyeloid lymphoid organs and present the antigen in order to activate Tcells. Immature DCs originate in the bone marrow and reside in theperiphery as immature cells.

Displaying: The process of localizing a peptide:antigen complex, or apeptide, on the outer surface of a cell where the peptide:antigencomplex or peptide is accessible to a second cell, molecules displayedby a second cell, or soluble factors. A peptide, or a peptide:antigencomplex, is “displayed” by a cell when it is present on the outersurface of the cell and is accessible to a second cell, to moleculesdisplayed by the second cell, or to soluble factors.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, e.g., that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide, such a Mycobacterium polypeptide.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (e.g., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5 or 3′ regions of the gene. Bothconstitutive and inducible promoters, are included (see e.g., Bitter etal., Meth. Enzymol. 153:516-544, 1987). For example, when cloning inbacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences. In one embodiment, thepromoter is a cytomegalovirus promoter.

Functionally Equivalent: Sequence alterations, such as in an epitope ofan antigen, which yield the same results as described herein. Suchsequence alterations can include, but are not limited to, conservativesubstitutions, deletions, mutations, frameshifts, and insertions.

Heterologous: Originating from separate genetic sources or species. Apolypeptide that is heterologous to an Mtb polypeptide originates from anucleic acid that does not encode the Mtb polypeptide or another Mtbpolypeptide. In one specific, non-limiting example, a polypeptidecomprising nine consecutive amino acids from an Mtb polypeptide, or atmost 20 consecutive amino acids from the Mtb polypeptide, and aheterologous amino acid sequence includes a β-galactosidase, a maltosebinding protein, and albumin, hepatitis B surface antigen, or animmunoglobulin amino acid sequence. Generally, an antibody thatspecifically binds to a protein of interest will not specifically bindto a heterologous protein.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The cell can bemammalian, such as a human cell. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used.

Human Leukocyte Antigen (HLA): A genetic designation of the human majorhistocompatibility complex (MHC). Individual loci are designated byuppercase letters, as in HLA-E, and alleles are designated by numbers,as in HLA-A*0201. The three main MHC class I genes are called HLA-A,HLA-B, and HLA-C. However, there are many genes that encode β2microglobulin-associated cell surface molecules that are linked to theMHC class I genes. The expression of these genes is variable, both inthe tissue distribution and the amount expressed on cells; these geneshave been termed the MHC class IB genes.

Immune response: A response of a cell of the immune system, such as a Bcell, natural killer cell, or a T cell, to a stimulus. In oneembodiment, the response is specific for a particular antigen (an“antigen-specific response”). In one embodiment, an immune response is aT cell response, such as a Th1, Th2, or Th3 response. In anotherembodiment, an immune response is a response of a suppressor T cell.

Immunogenic composition: A composition comprising an effective amount ofan immunogenic Mtb polypeptide or a nucleic acid encoding theimmunogenic Mtb polypeptide that induces a measurable T response againstMtb, such as a CD8⁺ T cell response, or induces a measurable B cellresponse (such as production of antibodies that specifically bind an Mtbpolypeptide). For in vitro use, the immunogenic composition can consistof the isolated nucleic acid, vector including the nucleic acid/orimmunogenic peptide. For in vivo use, the immunogenic composition willtypically comprise the nucleic acid, vector including the nucleic acid,and/or immunogenic polypeptide in pharmaceutically acceptable carriersand/or other agents. An immunogenic composition can optionally includean adjuvant, a costimulatory molecule, or a nucleic acid encoding acostimulatory molecule. An Mtb polypeptide, or nucleic acid encoding thepolypeptide, can be readily tested for its ability to induce a CD8⁺ Tcell response.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence such that the peptide will bind an MHC molecule andinduce a T cell response, such as a CD8⁺ or CD4⁺ T cell response, or a Bcell response (such as antibody production) against the antigen fromwhich the immunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide.

Immunogenic peptides can also be identified by measuring their bindingto a specific MHC protein and by their ability to stimulate CD4 and/orCD8 when presented in the context of the MHC protein. In one example, animmunogenic “Mtb peptide” is a series of contiguous amino acid residuesfrom the Mtb protein generally between 9 and 20 amino acids in length,such as about 8 to 11 residues in length.

Generally, immunogenic Mtb polypeptides can be used to induce an immuneresponse in a subject, such as a B cell response or a T cell response.In one example, an immunogenic Mtb polypeptide, when bound to a MHCClass I molecule, activates CD8⁺ T cells, such as cytotoxic Tlymphocytes (CTLs) against Mtb. Induction of CTLs using syntheticpeptides and CTL cytotoxicity assays are known in the art (see U.S. Pat.No. 5,662,907, which is incorporated herein by reference). In oneexample, an immunogenic peptide includes an allele-specific motif orother sequence such that the peptide will bind an MHC molecule andinduce a CD8⁺ response against the antigen from which the immunogenicpeptide is derived. A CD8⁺ T cell that specifically recognizes an Mtbpolypeptide is activated, proliferates, and/or secretes cytokines inresponse to that specific polypeptide, and not to other, non-relatedpolypeptides.

Inhibiting or treating a disease: Inhibiting a disease, such astuberculosis, refers to inhibiting the full development of a disease. Inseveral examples, inhibiting a disease refers to lessening symptoms of atuberculosis. “Treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionrelated to the disease, such as tuberculosis.

Interferon gamma (IFN-γ): IFN-γ is a dimeric protein with subunits of146 amino acids. The protein is glycosylated at two sites, and the pI is8.3-8.5. IFN-γ is synthesized as a precursor protein of 166 amino acidsincluding a secretory signal sequence of 23 amino acids. Two molecularforms of the biologically active protein of 20 and 25 kDa have beendescribed. Both of them are glycosylated at position 25. The 25 kDa formis also glycosylated at position 97. The observed differences of naturalIFN-γ with respect to molecular mass and charge are due to variableglycosylation patterns. 40-60 kDa forms observed under non-denaturingconditions are dimers and tetramers of IFN-γ. The human gene has alength of approximately 6 kb. It contains four exons and maps tochromosome 12q24.1.

IFN-γ can be detected by sensitive immunoassays, such as an ELSA testthat allows detection of individual cells producing IFN-γ. Minuteamounts of IFN-γ can be detected indirectly by measuring IFN-inducedproteins such as Mx protein. The induction of the synthesis of IP-10 hasalso been used to measure IFN-γ concentrations. In addition, bioassayscan be used to detect IFN-γ, such as an assay that employs induction ofindoleamine 2,3-dioxygenase activity in 2D9 cells. The production ofIFN-γ can be used to assess T cell activation, such as activation of a Tcell by a Mycobacterium antigen.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein, or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, such as otherchromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell, as well as chemically synthesized nucleicacids or proteins, or fragments thereof.

Linker sequence: A linker sequence is an amino acid sequence thatcovalently links two polypeptide domains. Linker sequences can beincluded in the between the Mtb epitopes disclosed herein to providerotational freedom to the linked polypeptide domains and thereby topromote proper domain folding and presentation to the MHC. By way ofexample, in a recombinant polypeptide comprising two Mtb domains, linkersequences can be provided between them, such as a polypeptide comprisingMtb polypeptide-linker-Mtb polypeptide. Linker sequences, which aregenerally between 2 and 25 amino acids in length, are well known in theart and include, but are not limited to, the glycine(4)-serine spacer(GGGGS ×3) described by Chaudhary et al., Nature 339:394-397, 1989.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells.

Mycobacteria: A genus of aerobic intracellular bacterial organisms. Uponinvasion of a host, these organisms survive within endosomalcompartments of monocytes and macrophages. Human mycobacterial diseasesinclude tuberculosis (cause by M. tuberculosis), Leprosy (caused by M.leprae), Bairnsdale ulcers (caused by M. ulcerans), and other infectionsthat can be caused by M. marinum, M kansasii, M. scrofulaceum, M.szulgai, M. xenopi, M. fortuitum, M. haemophilum, M. chelonei, and M.intracelluare. Mycobacterium strains that were previously considered tobe nonpathogenic (such as M. avium) are also now known to be majorkillers of immunosuppressed AIDS patients.

The major response to mycobacteria involves cell mediatedhypersensitivity (DTH) reactions with T cells and macrophages playingmajor roles in the intracellular killing and walling off (or containing)of the organism (granuloma formation). A major T cell response involvesCD4⁺ lymphocytes that recognize mycobacterial heat shock proteins andimmunodominant antigens.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, the openreading frames are aligned.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a polypeptide.

Peptide Modifications: Mycobacterium polypeptides include syntheticembodiments of peptides described herein. In addition, analogues(non-peptide organic molecules), derivatives (chemically functionalizedpeptide molecules obtained starting with the disclosed peptidesequences) and variants (homologs) of these proteins can be utilized inthe methods described herein. Each polypeptide of the invention iscomprised of a sequence of amino acids, which may be either L- and/orD-amino acids, naturally occurring and otherwise.

Peptides may be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpeptides, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring Amino groups of the peptide,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into the peptides of thisinvention to select and provide conformational constraints to thestructure that result in enhanced stability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe peptide backbone and component amino acid side chains, resulting insuch peptido- and organomimetics of a Mycobacterium polypeptide havingmeasurable or enhanced ability to generate an immune response. Forcomputer modeling applications, a pharmacophore is an idealized,three-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs,” in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press, Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptionsof techniques used in CADD. Also included are mimetics prepared usingsuch techniques.

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful with the polypeptides and nucleic acids described hereinare conventional. Remington's Pharmaceutical Sciences, by E. W. Martin,Mack Publishing Co., Easton, Pa., 19th Edition (1995), describescompositions and formulations suitable for pharmaceutical delivery ofthe polypeptides or polynucleotides herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polynucleotide: A linear nucleotide sequence, including sequences ofgreater than 100 nucleotide bases in length.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). A “peptide” is a chain of amino acids that is lessthan 100 amino acids in length. In one embodiment, a “peptide” is aportion of a polypeptide, such as about 8-11, 9-12, or about 10, 20, 30,40, 50, or 100 contiguous amino acids of a polypeptide that is greaterthan 100 amino acids in length.

Probes and primers: Nucleic acid probes and primers may readily beprepared based on the nucleic acids provided by this invention. A probecomprises an isolated nucleic acid attached to a detectable label orreporter molecule. Typical labels include radioactive isotopes, ligands,chemiluminescent agents, and enzymes. Methods for labeling and guidancein the choice of labels appropriate for various purposes are discussed,e.g., in Sambrook et al. (1989) and Ausubel et al. (1987).

Primers are short nucleic acids, preferably DNA oligonucleotides 15nucleotides or more in length. Primers may be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a DNA polymerase enzyme. Primer pairs canbe used for amplification of a nucleic acid sequence, e.g., by thepolymerase chain reaction (PCR) or other nucleic-acid amplificationmethods known in the art.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989, and Current Protocols in Molecular Biology, ed.Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1987(with periodic updates). PCR primer pairs can be derived from a knownsequence, for example, by using computer programs intended for thatpurpose such as Primer3 (Version 0.4.0, Whitehead Institute forBiomedical Research, Cambridge, Mass.).

Preventing or treating a disease: “Preventing” a disease refers toinhibiting the full development of a disease, for example in a personwho is known to be at risk of infection with M. tuberculosis or M.leprae. An example of a person with a known predisposition is someoneliving with a person diagnosed with tuberculosis, health careprofessionals, or someone who has been exposed to M. tuberculosis.“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition, such as tuberculosis,after it has begun to develop.

Promoter: A promoter is an array of nucleic acid control sequences whichdirect transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, such as, inthe case of a polymerase II type promoter, a TATA element. A promoteralso optionally includes distal enhancer or repressor elements which canbe located as much as several thousand base pairs from the start site oftranscription. The promoter can be a constitutive or an induciblepromoter. A specific, non-limiting example of a promoter is the HCMV IEpromoter.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified antigenpreparation is one in which the antigen is more pure than the protein inits originating environment within a cell. A preparation of an antigenis typically purified such that the antigen represents at least 50% ofthe total protein content of the preparation. However, more highlypurified preparations may be required for certain applications. Forexample, for such applications, preparations in which the antigencomprises at least 75% or at least 90% of the total protein content maybe employed.

Recombinant: A recombinant nucleic acid or polypeptide is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two or more otherwise separated segmentsof sequence. This artificial combination is often accomplished bychemical synthesis or, more commonly, by the artificial manipulation ofisolated segments of nucleic acids, e.g., by genetic engineeringtechniques.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Variants of antigen polypeptides will possess a relatively high degreeof sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Altschul et al. (1994) presents a detailed consideration ofsequence alignment methods and homology calculations. The NCBI BasicLocal Alignment Search Tool (BLAST) (Altschul et al., 1990) is availablefrom several sources, including the National Center for BiotechnologyInformation (NCBI, Bethesda, Md.) and on the Internet, for use inconnection with the sequence analysis programs blastp, blastn, blastx,tblastn and tblastx. It can be accessed at the NCBI website. Adescription of how to determine sequence identity using this program isavailable at the NCBI website, as are the default parameters.

Variants of antigenic polypeptides, such as a Mycobacterium polypeptide,are typically characterized by possession of at least 50% sequenceidentity counted over the full length alignment with the amino acidsequence of a native antigen sequence using the NCBI Blast 2.0, gappedblastp set to default parameters. Proteins with even greater similarityto the reference sequences will show increasing percentage identitieswhen assessed by this method, such as at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 90% or at least 95%sequence identity. When less than the entire sequence is being comparedfor sequence identity, variants will typically possess at least 75%sequence identity over short windows of 10-20 amino acids, and maypossess sequence identities of at least 85% or at least 90% or 95%depending on their similarity to the reference sequence. Methods fordetermining sequence identity over such short windows are described atthe NCBI website.

Therapeutically active polypeptide: An agent, such as an epitope of Mtbthat causes induction of an immune response, as measured by clinicalresponse (for example increase in a population of immune cells,increased cytolytic activity against Mtb, or measurable reduction of asymptom of an infection). Therapeutically active molecules can also bemade from nucleic acids. Examples of a nucleic acid basedtherapeutically active molecule is a nucleic acid sequence that encodesan Mtb epitope, wherein the nucleic acid sequence is operably linked toa control element such as a promoter.

In one embodiment, a therapeutically effective amount of an Mtbpolypeptide is an amount used to generate an immune response. In severalexamples, “treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of tuberculosis.

Therapeutically effective amount: A dose sufficient to inhibitadvancement, or to cause regression of the disease, or which is capableof relieving symptoms caused by the disease. In one embodiment, atherapeutically effective dose is a dose sufficient to inhibit orprevent advancement or relieve symptoms of tuberculosis.

Transduced and Transformed: A virus or vector “transduces” a cell whenit transfers nucleic acid into the cell. A cell is “transformed” by anucleic acid transduced into the cell when the DNA becomes stablyreplicated by the cell, either by incorporation of the nucleic acid intothe cellular genome, or by episomal replication. As used herein, theterm transformation encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Tuberculosis (TB): A disease that is generally caused by Mycobacteriumtuberculosis that usually infects the lungs. However, other “atypical”mycobacteria such as M. kansasii may produce a similar clinical andpathologic appearance of disease.

Transmission of M. tuberculosis occurs by the airborne route in confinedareas with poor ventilation. In more than 90% of cases, followinginfection with M. tuberculosis, the immune system prevents developmentof disease from M. tuberculosis, often called, active tuberculosis.However, not all of the M. tuberculosis is killed, and thus tiny, hardcapsules are formed. “Primary tuberculosis” is seen as disease thatdevelops following an initial infection, usually in children. Theinitial focus of infection is a small subpleural granuloma accompaniedby granulomatous hilar lymph node infection. Together, these make up theGhon complex. In nearly all cases, these granulomas resolve and there isno further spread of the infection. “Secondary tuberculosis” is seenmostly in adults as a reactivation of previous infection (orreinfection), particularly when health status declines. Thegranulomatous inflammation is much more florid and widespread.Typically, the upper lung lobes are most affected, and cavitation canoccur. Dissemination of tuberculosis outside of the lungs can lead tothe appearance of a number of uncommon findings with characteristicpatterns that include skeletal tuberculosis, genital tract tuberculosis,urinary tract tuberculosis, central nervous system (CNS) tuberculosis,gastrointestinal tuberculosis, adrenal tuberculosis, scrofula, andcardiac tuberculosis. “Latent” tuberculosis is an Mtb infection in anindividual that can be detected by a diagnostic assay, such as, but notlimited to a tuberculin skin test (TST) wherein the infection does notproduce symptoms in that individual. “Active” tuberculosis is asymptomatic Mtb infection in a subject.

Microscopically, the inflammation produced with TB infection isgranulomatous, with epithelioid macrophages and Langhans giant cellsalong with lymphocytes, plasma cells, maybe a few polymorphonuclearcells, fibroblasts with collagen, and characteristic caseous necrosis inthe center. The inflammatory response is mediated by a type IVhypersensitivity reaction, and skin testing is based on this reaction.In some examples, tuberculosis can be diagnosed by a skin test, an acidfast stain, an auramine stain, or a combination thereof. The most commonspecimen screened is sputum, but the histologic stains can also beperformed on tissues or other body fluids.

TB is a frequent complication of HIV infection. TB infection in subjectsinfected with a human immunodeficiency virus (HIV) can spread readilyand progress rapidly to active disease. Specific symptoms of lungdisease due to Mtb infection include chronic cough and spitting blood.Other symptoms of TB disease include fatigue, loss of appetite, weightloss, fever and drenching night sweats.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergene and other genetic elements known in the art. Vectors includeplasmid vectors, including plasmids for expression in gram negative andgram positive bacterial cell. Exemplary vectors include those forexpression in E. coli and Salmonella. Vectors also include viralvectors, such as, but not limited to, retrovirus, orthopox, avipox,fowlpox, capripox, suipox, adenovirus, herpes virus, alpha virus,baculovirus, Sindbis virus, vaccinia virus, and poliovirus vectors.Vectors also include vectors for expression in yeast cells

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

III. MYCOBACTERIUM POLYPEPTIDES

It is disclosed herein that several Mycobacterium polypeptides can beused to induce an immune response to Mtb, such as a T cell response. Inseveral embodiments, the polypeptide comprises or consists of the aminoacid sequence set forth as:

VPHPWDTGDHERNWQGYFIPAMSVLRNRVGARTHAELRDAENDLVEARVIELREDPNLLGDRTDLAYLRAIHRQLFQDIYVWAGDLRTVGIEKEDESFCAPGGISRPMEHVAAEIYQLDRLRAVGEGDLAGQVAYRYDYVNYAHPFREGNGRSTREFFDLLLSERGSGLDWGKTDLEELHGACHVARANSDLTGLVAMFKGILDAEPTYDF (SEQ ID NO: 1; see also TUBERCULIST No. Rv3641c, as available on Jun. 8, 2009, incorporated herein by reference, known as fic).MDFALLPPEVNSARMYTGPGAGSLLAAAGGWDSLAAELATTAEAYGSVLSGLAALHWRGPAAESMAVTAAPYIGWLYTTAEKTQQTAIQARAAALAFEQAYAMTLPPPVVAANRIQLLALIATNFFGQNTAAIAATEAQYAEMWAQDAAAMYGYATASAAAALLTPFSPPRQTTNPAGLTAQAAAVSQATDPLSLLIETVTQALQALTIPSFIPEDFTFLDAIFAGYATVGVTQDVESFVAGTIGAESNL GLLNVGDENPAEVTPGDFGIGELVSATSPGGGVSASGAGGAASVGNTVLA SVGRANSIGQLSVPPSWAAPSTRPVSALSPAGLTTLPGTDVAEHGMPGVPGVPVAAGRASGVLPRYGVRLTVMAHPPAAG (SEQ ID NO: 2; seealso TUBERCULIST No. Ry3136, as available on Jun.8, 2009, incorporated herein by reference, known as PPE51 or PPE).MTEPRPVFAVVISAGLSAIPMVGGPLQTVFDAIEERTRHRAETTTREICESVGGADTVLSRIDKNPELEPLLSQAIEAATRTSMEAKRRLLAQAAAAALEDDQKVEPASLIVATLSQLEPVHIHALVRLAKAAKSSPDQDEIQRREVMRAASKVEPVPVLAALIQTGVAIATTTVWHGNGTGTPAEESGHILIHDVSDFGHRLLAYLRAADAGAELLILPSGGSAPTGDHPTPHPSTSR (SEQ ID NO: 3; see also TUBERCULIST No. Rv0394c, asavailable on Jun. 8, 2009, incorporated herein by reference).MADFLTLSPEVNSARMYAGGGPGSLSAAAAAWDELAAELWLAAASFESVCSGLADRWWQGPSSRMMAAQAARHTGWLAAAATQAEGAASQAQTMALAYEAAFAATVHPALVAANRALVAWLAGSNVFGQNTPAIAAAEAIYEQMWAQDVVAMLNYHAVASAVGARLRPWQQLLHELPRRLGGEHSDSTNTELANPSSTTTRITVPGASPVHAATLLPFIGRLLAARYAELNTAIGTNWFPGTTPEVVSYPATIGVLSGSLGAVDANQSIAIGQQMLHNEILAATASGQPVTVAGLSMGSMVIDRELAYLAIDPNAPPSSALTFVELAGPERGLAQTYLPVGTTIPIAGYTVGNAPESQYNTSVVYSQYDIVVADPPDRPWNLLAGANALMGAAYFHDLTAYAAPQQGIEIAAVTSSLGGTTTTYMIPSPTLPLLLPLKQIGVPDWIVGGLNNVLKPLVDAGYSQYAPTAGPYFSHGNLVW (SEQ ID NO:4; see also TUBERCULIST No. Rv3539, as availableon Jun. 8, 2009, incorporated herein by reference,known as PPE63 or PPE).MTLDVPVNQGHVPPGSVACCLVGVTAVADGIAGHSLSNFGALPPEINSGRMYSGPGSGPLMAAAAAWDGLAAELSSAATGYGAAISELTNMRWWSGPASDSMVAAVLPFVGWLSTTATLAEQAAMQARAAAAAFEAAFAMTVPPPAIAANRTLLMTLVDTNWFGQNTPAIATTESQYAEMWAQDAAAMYGYASAAAPATVLTPFAPPPQTTNATGLVGHATAVAALRGQHSWAAAIPWSDIQKYWMMFLGALATAEGFIYDSGGLTLNALQFVGGMLWSTALAEAGAAEAAAGAGGAAGWSAWSQLGAGPVAASATLAAKIGPMSVPPGWSAPPATPQAQTVARSIPGIRSAAEAAETSVLLRGAPTPGRSRAAHMGRRYGRRLTVMADRPNVG(SEQ ID NO: 5; see also TUBERCULIST No. Rv1706c,as available on Jun. 8, 2009, incorporated hereinby reference, known as PPE23 or PPE).MDFGALPPEINSARMYAGAGAGPMMAAGAAWNGLAAELGTTAASYESVITRLTTESWMGPASMAMVAAAQPYLAWLTYTAEAAAHAGSQAMASAAAYEAAYAMTVPPEVVAANRALLAALVATNVLGINTPAIMATEALYAEMWAQDALAMYGYAAASGAAGMLQPLSPPSQTTNPGGLAAQSAAVGSAAATAAVNQVSVADLISSLPNAVSGLASPVTSVLDSTGLSGIIADIDALLATPFVAN IINSAVNTAAWYVNAAIPTAIFLANALNSGAPVAIAEGAIEAAEGAASAAAAGLADSVTPAGLGASLGEATLVGRLSVPAAWSTAAPATTAGATALEGSGWTVAAEEAGPVTGMMPGMASAAKGTGAYAGPRYGFKPTVMPKQVVV (SEQ ID NO: 6; see also TUBERCULIST No. Rv1039c,as available on Jun. 8, 2009, incorporated hereinby reference, known as PPE15 or PPE).MAHFSVLPPEINSLRMYLGAGSAPMLQAAAAWDGLAAELGTAASSFSSVTTGLTGQAWQGPASAAMAAAAAPYAGFLTTASAQAQLAAGQAKAVASVFEAAKAAIVPPAAVAANREAFLALIRSNWLGLNAPWIAAVESLYEEYWAADVAAMTGYHAGASQAAAQLPLPAGLQQFLNTLPNLGIGNQGNANLGGGNTGSGNIGNGNKGSSNLGGGNIGNNNIGSGNRGSDNFGAGNVGTGNIGFGNQGPIDVNLLATPGQNNVGLGNIGNNNMGFGNTGDANTGGGNTGNGNIGGGNTGNNNFGFGNTGNNNIGIGLTGNNQMGINLAGLLNSGSGNIGIGNSGTNNIGLFNSGSGNIGVFNTGANTLVPGDLNNLGVGNSGNANIGFGNAGVLNTGFGNASILNTGLGNAGELNTGFGNAGFVNTGFDNSGNVNTGNGNSGNINTGSWNAGNVNTGFGIITDSGLTNSGFGNTGTDVSGFFNTPTGPLAVDVSGFFNTASGGTVINGQTSGIGNIGVPGTLFGSVRSGLNTGLFNMGTAISGLFNLRQLLG (SEQ ID NO: 7; see also TUBERCULIST No. Rv3558, as available on Jun. 8, 2009, incorporatedherein by reference, known as PPE64 or PPE).MEYLIAAQDVLVAAAADLEGIGSALAAANRAAEAPTTGLLAAGADEVSAAIASLFSGNAQAYQALSAQAAAFHQQFVRALSSAAGSYAAAEAANASPMQAVLDVVNGPTQLLLGRPLIGDGANGGPGQNGGDGGLLYGNGGNGGSSSTPGQPGGRGGAAGLIGNGGAGGAGGPGANGGAGGNGGWLYGNGGLGGNGGAATQIGGNGGNGGHGGNAGLWGNGGAGGAGAAGAAGANGQNPVSHQVTHATDGADGTTGPDGNGTDAGSGSNAVNPGVGGGAGGIGGDGTNLGQTDVSGGAGGDGGDGANFASGGAGGNGGAAQSGFGDAVGGNGGAGGNGGAGGGGGLGGAGGSANVANAGNSIGGNGGAGGNGGIGAPGGAGGAGGNANQDNPPGGNSTGGNGGAGGDGGVGASADVGGAGGFGGSGGRGGLLLGTGGAGGDGGVGGDGGIGAQGGSGGNGGNGGIGADGMANQDGDGGDGGNGGDGGAGGAGGVGGNGGTGGAGGLFGQSGSPGSGAAGGLGGAGGNGGAGGGGGTGFNPGAPGDPGTQGATGANGQHGLN (SEQ ID NO: 8; seealso TUBERCULIST No. Rv1243c, as available onOct. 6, 2009; incorporated herein by reference, known as PE_PGRS23).MVMSLMVAPELVAAAAADLTGIGQAISAANAAAAGPTTQVLAAAGDEVSAAIAALFGTHAQEYQALSARVATFHEQFVRSLTAAGSAYATAEAANASPLQALEQQVLGAINAPTQLWLGRPLIGDGVHGAPGTGQPGGAGGLLWGNGGNGGSGAAGQVGGPGGAAGLFGNGGSGGSGGAGAAGGVGGSGGWLNGNGGAGGAGGTGANGGAGGNAWLFGAGGSGGAGTNGGVGGSGGFVYGNGGAGGIGGIGGIGGNGGDAGLFGNGGAGGAGAAGLPGAAGLNGGDGSDGGNGGTGGNGGRGGLLVGNGGAGGAGGVGGDGGKGGAGDPSFAVNNGAGGNGGHGGNPGVGGAGGAGGLLAGAHGAAGATPTSGGNGGDGGIGATANSPLQAGGAGGNGGHGGLVGNGGTGGAGGAGHAGSTGATGTALQPTGGNGTNGGAGGHGGNGGNGGAQHGDGGVGGKGGAGGSGGAGGNGFDAATLGSPGADGGMGGNGGKGGDGGKAGDGGAGAAGDVTLAVNQGAGGDGGNGGEVGVGGKGGAGGVSANPALNGSAGANGTAPTSGGNGGNGGAGATPTVAGENGGAGGNGGHGGSVGNGGAGGAGGNGVAGTGLALNGGNGGNGGIGGNGGSAAGTGGDGGKGGNGGAGANGQDFSASANGANGGQGGNGGNGGIGGKGGDAFATFAKAGNGGAGGNGGNVGVAGQGGAGGKGAIPAMKGATGADGTAPTSGGDGGNGGNGASPTVAGGNGGDGGKGGSGGNVGNGGNGGAGGNGAAGQAGTPGPTSGDSGTSGTDGGAGGNGGAGGAGGTLAGHGGNGGKGGNGGQGGIGGAGERGADGAGPNANGANGENGGSGGNGGDGGAGGNGGAGGKAQAAGYTDGATGTGGDGGNGGDGGKAGDGGAGENGLNSGAMLPGGGTVGNPGTGGNGGNGGNAGVGGTGGKAGTGSLTGLDGTDGITPNGGNGGNGGNGGKGGTAGNGSGAAGGNGGNGGSGLNGGDAGNGGNGGGALNQAGFFGTGGKGGNGGNGGAGMINGGLGGFGGAGGGGAVDVAATTGGAGGNGGAGGFASTGLGGPGGAGGPGGAGDFASGVGGVGGAGGDGGAGGVGGFGGQGGIGGEGRTGGNGGSGGDGGGGISLGGNGGLGGNGGVSETGFGGAGGNGGYGGPGGPEGNGGLGGNGGAGGNGGVSTTGGDGGAGGKGGNGGDGGNVGLGGDAGSGGAGGNGGIGTDAGGAGGAGGAGGNGGSSKSTTTGNAGSGGAGGNGGTGLNGAGGAGGAGGNAGVAGVSFGNAVGGDGGNGGNGGHGGDGTTGGAGGKGGNGSSGAASGSGVVNVTAGHGGNGGNGGNGGNGSAGAGGQGGAGGSAGNGGHGGGATGGDGGNGGNGGNSGNSTGVAGLAGGAAGAGGNGGGTSSAAGHGGSGGSGGSGTTGGAGAAGGNGGAGAGGGSLSTGQSGGPRRQRWCRWQRRRWLGRQRRRRWCRWQRRCRRQRWRWRCRQRRLRRQWRQGRRRCRPWLHRRRGRQGRRWRQRRFQQRQRSRWQRR (SEQID NO: 9; see also TUBERCULIST No. Rv3345c, asavailable on Oct. 6, 2009; incorporated herein byreference, known as PE_PGRS50).VIQTCEVELRWRASQLTLAIATCAGVALAAAVVAGRWQLIAFAAPLLGVLCSISWQRPVPVIQVHGDPDSQRCFENEHVRVTVWVTTESVDAAVELTVSALAGMQFEALESVSRRTTTVSAVAQRWGRYPIRARVAVVARGGLLMGAGTVDAAEIVVFPLTPPQSTPLPQTELLDRLGAHLTRHVGPGVEYADIRPYVPGDQLRAVNWVVSARRGRLHVTRRLTDRAADVVVLIDMYRQPAGPATEATERVVRGAAQVVQTALRNGDRAGIVALGGNRPRWLGADIGQRQFYRVLDTVLGAGEGFENTTGTLAPRAAVPAGAVVIAFSTLLDTEFALALIDLRKRGHVVVAVDVLDSCPLQDQLDPLVVRMWALQRSAMYRDMATIGVDVLSWPADHSLQQSMGALPNRRRRGRGRASRARLP (SEQ ID NO: 10; see alsoTUBERCULIST No. Rv3163c, as available on Oct.6,2009; incorporated herein by reference).VNRRILTLMVALVPIVVFGVLLAVVTVPFVALGPGPTFDTLGEIDGKQVVQIVGTQTYPTSGHLNMTTVSQRDGLTLGEALALWLSGQEQLMPRDLVYPPGKSREEIENDNAADFKRSEAAAEYAALGYLKYPKAVTVASVMDPGPSVDKLQAGDAIDAVDGTPVGNLDQFTALLKNTKPGQEVTIDFRRKNEPPGIAQITLGKNKDRDQGVLGIEVVDAPWAPFAVDFHLANVGGPSAGLMFSLAVVDKLTSGHLVGSTFVAGTGTIAVDGKVGQIGGITHKMAAARAAGATVFLVPAKNCYEASSDSPPGLKLVKVETLSQAVDALHAMTSGSPTPSC (SEQ IDNO: 11; see also TUBERCULIST No. Rv3194c, asavailable on Oct. 6, 2009; incorporated herein by reference).MSFVVTAPPVLASAASDLGGIASMISEANAMAAVRTTALAPAAADEVSAAIAALFSSYARDYQTLSVQVTAFHVQFAQTLTNAGQLYAVVDVGNGVLLKTEQQVLGVINAPTQTLVGRPLIGDGTHGAPGTGQNGGAGGILWGNGGNGGSGAPGQPGGRGGDAGLFGHGGHGGVGGPGIAGAAGTAGLPGGNGANGGSGGIGGAGGAGGNGGLLFGNGGAGGQGGSGGLGGSGGTGGAGMAAGPAGGTGGIGGIGGIGGAGGVGGHGSALFGHGGINGDGGTGGMGGQGGAGGNGWAAEGITVGIGEQGGQGGDGGAGGAGGIGGSAGGIGGSQGAGGHGGDGGQGGAGGSGGVGGGGAGAGGDGGAGGIGGTGGNGSIGGAAGNGGNGGRGGAGGMATAGSDGGNGGGGGNGGVGVGSAGGAGGTGGDGGAAGAGGAPGHGYFQQPAPQGLPIGTGGTGGEGGAGGAGGDGGQGDIGFDGGRGGDGGPGGGGGAGGDGSGTFNAQANNGGDGGAGGVGGAGGTGGTGGVGADGGRGGDSGRGGDGGNAGHGGAAQFSGRGAYGGEGGSGGAGGNAGGAGTGGTAGSGGAGGFGGNGADGGNGGNGGNGGFGGINGTFGTNGAGGTGGLGTLLGGHNGNIGLNGATGGIGSTTLTNATVPLQLVNTTEPVVFISLNGGQMVPVLLDTGSTGLVMDSQFLTQNFGPVIGTGTAGYAGGLTYNYNTYSTTVDFGNGLLTLPTSVNVVTSSSPGTLGNFLSRSGAVGVLGIGPNNGFPGTSSIVTAMPGLLNNGVLIDESAGILQFGPNTLTGGITISGAPISTVAVQIDNGPLQQAPVMFDSGGINGTIPSALASLPSGGFVPAGTTISVYTSDGQTLLYSYTTTATNTPFVTSGGVMNTGHVPFAQQPIYVSYSPTAIGTTTFN (SEQ IDNO: 12; see also TUBERCULIST No. Rv0977, asavailable on Oct. 6, 2009; incorporated herein by reference).MTHDHAHSRGVPAMIKEIFAPHSHDAADSVDDTLESTAAGIRTVKISLLVLGLTALIQIVIVVMSGSVALAADTIHNFADALTAVPLWIAFALGAKPATRRYTYGFGRVEDLAGSFVVAMITMSAIIAGYEAIARLIHPQQIEHVGWVALAGLVGFIGNEWVALYRIRVGHRIGSAALIADGLHARTDGFTSLAVLCSAGGVALGFPLADPIVGLLITAAILAVLRTAARDVFRRLLDGVDPAMVDAAEQALAARPGVQAVRSVRMRWIGHRLHADAELDVDPALDLAQAHRIAHDAEHELTHTVPKLTTALIHAYPAEHGSSIPDRGRTVE (SEQ ID NO: 13;see also TUBERCULIST No. Rv2025c, as available onOct. 6, 2009; incorporated herein by reference).VVNFSVLPPEINSGRMFFGAGSGPMLAAAAAWDGLAAELGLAAESFGLVTSGLAGGSGQAWQGAAAAAMVVAAAPYAGWLAAAAARAGGAAVQAKAVAGAFEAARAAMVDPVVVAANRSAFVQLVLSNVFGQNAPAIAAAEATYEQMWAADVAAMVGYHGGASAAAAALAPWQQAVPGLSGLLGGAANAPAAAAQGAAQGLAELTLNLGVGNIGSLNLGSGNIGGTNVGSGNVGGTNLGSGNYGSLNWGSGNTGTGNAGSGNTGDYNPGSGNFGSGNFGSGNIGSLNVGSGNFGTLNLANGNNGDVNFGGGNTGDFNFGGGNNGTLNFGFGNTGSGNFGFGNTGNNNIGIGLTGDGQIGIGGLNSGTGNIGFGNSGNNNIGFFNSGDGNIGFFNSGDGNTGFGNAGNINTGFVVNAGNLNTGFGSAGNGNVGIFDGGNSNSGSFNVGFQNTGFGNSGAGNTGFFNAGDSNTGFANAGNVNTGFFNGGDINTGGFNGGNVNTGFGSALTQAGANSGFGNLGTGNSGWGNSDPSGTGNSGFFNTGNGNSGFSNAGPAMLPGFNSGFANIGSFNAGIANSGNNLAGISNSGDDSSGAVNSGSQNSGAFNAGVGLSGFFR(SEQ ID NO: 14; see alsoTUBERCULIST No. Rv2356c, as available on Oct. 6,2009; incorporated herein by reference, known as PPE40).MNYSVLPPEINSLRMFTGAGSAPMLAASVAWDRLAAELAVAASSFGSVTSGLAGQSWQGAAAAAMAAAAAPYAGWLAAAAARAAGASAQAKAVASAFEAARAATVHPMLVAANRNAFVQLVLSNLFGQNAPAIAAAEAMYEQMWAADVAAMVGYHGGASAAAAQLSSWSIGLQQALPAAPSALAAAIGLGNIGVGNLGGGNTGDYNLGSGNSGNANVGSGNSGNANVGSGNDGATNLGSGNIGNTNLGSGNVGNVNLGSGNRGFGNLGNGNFGSGNLGSGNTGSTNFGGGNLGSFNLGSGNIGSSNIGFGNNGDNNLGLGNNGNNNIGFGLTGDNLVGIGALNSGIGNLGFGNSGNNNIGFFNSGNNNVGFFNSGNNNFGFGNAGDINTGFGNAGDTNTGFGNAGFFNMGIGNAGNEDMGVGNGGSFNVGVGNAGNQSVGFGNAGTLNVGFANAGSINTGFANSGSINTGGFDSGDRNTGFGSSVDQSVSSSGFGNTGMNSSGFFNTGNVSAGYGNNGDVQSGINNTNSGGFNVGFYNSGAGTVGIANSGLQTTGIANSGTLNTGVANTGDHSSGGFNQGSDQSGFFGQP (SEQ ID NO: 15; see also TUBERCULIST No. Rv3159c,as available on Oct. 6, 2009; incorporated hereinby reference, known as PPE53).MSFVFAAPEALAAAAADMAGIGSTLNAANVVAAVPTTGVLAAAADEVSTQVAALLSAHAQGYQQLSRQMMTAFHDQFVQALRASADAYATAEASAAQTMVNAVNAPARALLGHPLISADASTGGGSNALSRVQSMFLGTGGSSALGGSAAANAAASGALQLQPTGGASGLSAVGALLPRAGAAAAAALPALAAESIGNAIKNLYNAVEPWVQYGFNLTAWAVGWLPYIGILAPQINFFYYLGEPIVQAVLFNAIDFVDGTVTFSQALTNIETATAASINQFINTEINWIRGFLPPLPPISPPGFPSLP (SEQ ID NO: 16; see also TUBERCULISTNo. Rv1172c, as available on Oct. 6, 2009;incorporated herein by reference, known as PE12).MDYAFLPPEINSARMYSGPGPNSMLVAAASWDALAAELASAAENYGSVIARLTGMHWWGPASTSMLAMS APYVEWLERTAAQTKQTATQARAAAAAFEQAHAMTVPPALVTGIRGAIVVETASASNTAGTPP (SEQ ID NO: 17;see also TUBERCULIST No. Rv3135, as available onJun. 8, 2009, incorporated herein by reference, known as PPE50 or PPE).LSASVSATTAHHGLPAHEVVLLLESDPYHGLSDGEAAQRLERFGPNTLAVVTRASLLARILRQFHHPLIYVLLVAGTITAGLKEFVDAAVIFGVVVINAIVGFIQESKAEAALQGLRSMVHTHAKVVREGHEHTMPSEELVPGDLVLLAAGDKVPADLRLVRQTGLSVNESALTGESTPVHKDEVALPEGTPVADRRNIAYSGTLVTAGHGAGIVVATGAETELGEIHRLVGAAEVVATPLTAKLAWFSKFLTIAILGLAALTFGVGLLRRQDAVETFTAAIALAVGAIPEGLPTAVTITLAIGMARMAKRRAVIRRLPAVETLGSTTVICADKTGTLTENQMTVQSIWTPHGEIRATGTGYAPDVLLCDTDDAPVPVNANAALRWSLLAGACSNDAALVRDGTRWQIVGDPTEGAMLVVAAKAGFNPERLATTLPQVAAIPFSSERQYMATLHRDGTDHVVLAKGAVERMLDLCGTEMGADGALRPLDRATVLRATEMLTSRGLRVLATGMGAGAGTPDDFDENVIPGSLALTGLQAMSDPPRAAAASAVAACHSAGIAVKMITGDHAGTATAIATEVGLLDNTEPAAGSVLTGAELAALSADQYPEAVDTASVFARVSPEQKLRLVQALQARGHVVAMTGDGVNDAPALRQANIGVAMGRGGTEVAKDAADMVLTDDDFATIEAAVEEGRGVFDNLTKFITWTLPTNLGEGLVILAAIAVGVALPILPTQILWINMTTAIALGLMLAFEPKEAGIMTRPPRDPDQPLLTGWLVRRTLLVSTLLVASAWWLFAWELDNGAGLHEARTAALNLFVVVEAFYLFSCRSLTRSAWRLGMFANRWIILGVSAQAIAQFAITYLPAMNMVFDTAPIDIGVWVRIFAVATAITIVVATDTLLPRIRAQPP (SEQ ID NO: 18; see also TUBERCULIST No.Rv1997, as available on June 8, 2009, incorporatedherein by reference, known as ctpF).

In a second embodiment, an Mtb polypeptide of use in the methodsdisclosed herein has a sequence at least 75%, 85%, 90%, 95%, 96%, 97%,98% or 99% homologous to the amino acid sequence set forth in one of SEQID NOs: 1-18. For example, the polypeptide can have an amino acidsequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to oneof the amino acid sequences set forth in SEQ ID NOs: 1-18. Exemplarysequences can be obtained using computer programs that are readilyavailable on the internet and the amino acid sequences set forth herein.In one example, the polypeptide retains a function of the Mtb protein,such as binding to an antibody that specifically binds the Mtb epitope.

Minor modifications of an Mtb polypeptide primary amino acid sequencesmay result in peptides which have substantially equivalent activity ascompared to the unmodified counterpart polypeptide described herein.Such modifications may be deliberate, as by site-directed mutagenesis,or may be spontaneous. All of the polypeptides produced by thesemodifications are included herein. Thus, a specific, non-limitingexample of an Mtb polypeptide is a conservative variant of the Mtbpolypeptide (such as a single conservative amino acid substitution, forexample, one or more conservative amino acid substitutions, for example1-10 conservative substitutions, 2-5 conservative substitutions, 4-9conservative substitutions, such as 1, 2, 5 or 10 conservativesubstitutions). A table of conservative substitutions is providedherein. Substitutions of the amino acids sequence shown in SEQ ID NOs:1-18 can be made based on this table.

Mtb polypeptides are disclosed herein that can be used to induce animmune response to Mtb. These peptides include or consist of at leastnine amino acids, such as nine to twenty amino acids consecutive aminoacids of an Mtb polypeptide set forth above. In particular non-limitingexamples, the Mtb polypeptide includes or consists of MDFALLPPEVNSARM(amino acids 1-15 of SEQ ID NO: 2) or AEMWAQDAA (amino acids 141-149 ofSEQ ID NO: 2). Specific, non-limiting examples are fifteen, fourteen,thirteen, twelve, eleven, ten, or nine consecutive amino acids of one ofthe Mtb polypeptides set forth above. In these examples, the Mtbpolypeptide does not include the full-length amino acid sequences setforth as SEQ ID NOs: 1-18.

In several embodiments, the isolated Mtb polypeptide is included in afusion protein. Thus, the fusion protein can include the Mtb polypeptide(see above) and a second heterologous moiety, such as a myc protein, anenzyme or a carrier (such as a hepatitis carrier protein or bovine serumalbumin) covalently linked to the Mtb polypeptide. In several examples,a polypeptide consisting of nine to twelve amino acids of one of theamino acid sequences set forth as SEQ ID NOs: 1-18 that bind MHC class Iis covalently linked to a carrier. In an additional example, apolypeptide consisting of one of the amino acid sequences set forth asone of SEQ ID NOs: 1-18 is covalently linked to a carrier.

In additional examples, the polypeptide can be a fusion protein and canalso include heterologous sequences to Mtb. Thus, in several specificnon-limiting examples, the immunogenic peptide is a fusion polypeptide,for example the polypeptide includes six sequential histidine residues,a β-galactosidase amino acid sequence, or an immunoglobulin amino acidsequence. The polypeptide can also be covalently linked to a carrier. Inadditional embodiments, the protein consists of the Mtb polypeptide.

The polypeptide can optionally include repetitions of one or more of theMtb polypeptides disclosed herein. In one specific, non-limitingexample, the polypeptide includes two, three, four, five, or up to tenrepetitions of one of the Mtb polypeptides described above.Alternatively, more than one polypeptide can be included in a fusionpolypeptide. Thus, in several examples, the polypeptide can include atleast two, at least three, at least four, at least five or at least sixof the amino acid sequences set forth as SEQ ID NOs: 1-18 or nine totwenty amino acids of one of the amino acid sequences set forth as SEQID NOs: 1-18 and repetitions of these sequences. A linker sequence canoptionally be included between the Mtb polypeptides.

The Mtb polypeptides disclosed herein can be chemically synthesized bystandard methods, or can be produced recombinantly. An exemplary processfor polypeptide production is described in Lu et al., FEBS Lett.429:31-35, 1998. They can also be isolated by methods includingpreparative chromatography and immunological separations. Polypeptidescan also be produced using molecular genetic techniques, such as byinserting a nucleic acid encoding Mtb or an epitope thereof into anexpression vector, introducing the expression vector into a host cell,and isolating the polypeptide (see below).

In particular embodiments provided herein, one or more of the disclosedMtb polypeptides (or fragments thereof) can be conjugated to a substrateor solid support, such as a plate or array. In one example, the plate orarray includes, consists essentially of, or consists of one (such as 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all) of SEQ IDNOs: 1-18 or fragments thereof. In some examples, the plate or arrayalso includes one or more control polypeptides. Methods for selecting anappropriate substrate and constructing a plate or array are well knownto one of skill in the art (see, e.g., U.S. Pat. Nos. 5,143,854;5,405,783; 5,445,934; and 5,744,305; all incorporated herein byreference).

Polynucleotides encoding the Mtb polypeptides disclosed herein are alsoprovided. Exemplary nucleic acid sequences are set forth as SEQ ID NOs:19-36. These polynucleotides include DNA, cDNA and RNA sequences whichencode the polypeptide of interest. Silent mutations in the codingsequence result from the degeneracy (i.e., redundancy) of the geneticcode, whereby more than one codon can encode the same amino acidresidue. Tables showing the standard genetic code can be found invarious sources (e.g., L. Stryer, 1988, Biochemistry, 3^(rd) Edition,W.H. Freeman and Co., NY).

A nucleic acid encoding an Mtb polypeptide can be cloned or amplified byin vitro methods, such as the polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR) andthe Qβ replicase amplification system (QB). For example, apolynucleotide encoding the protein can be isolated by polymerase chainreaction of cDNA using primers based on the DNA sequence of themolecule. A wide variety of cloning and in vitro amplificationmethodologies are well known to persons skilled in the art. PCR methodsare described in, for example, U.S. Pat. No. 4,683,195; Mullis et al.,Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; and Erlich, ed., PCRTechnology, (Stockton Press, N Y, 1989). Polynucleotides also can beisolated by screening genomic or cDNA libraries with probes selectedfrom the sequences of the desired polynucleotide under stringenthybridization conditions.

The polynucleotides encoding an Mtb polypeptide include a recombinantDNA which is incorporated into a vector into an autonomously replicatingplasmid or virus or into the genomic DNA of a prokaryote or eukaryote,or which exists as a separate molecule (such as a cDNA) independent ofother sequences. The nucleotides of the invention can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single and double forms of DNA.

In one embodiment, vectors are used for expression in yeast such as S.cerevisiae or Kluyveromyces lactis. Several promoters are known to be ofuse in yeast expression systems such as the constitutive promotersplasma membrane H⁺-ATPase (PMA1), glyceraldehyde-3-phosphatedehydrogenase (GPD), phosphoglycerate kinase-1 (PGK1), alcoholdehydrogenase-1 (ADH1), and pleiotropic drug-resistant pump (PDR5). Inaddition, many inducible promoters are of use, such as GAL1-10 (inducedby galactose), PHO5 (induced by low extracellular inorganic phosphate),and tandem heat shock HSE elements (induced by temperature elevation to37° C.). Promoters that direct variable expression in response to atitratable inducer include the methionine-responsive MET3 and MET25promoters and copper-dependent CUP1 promoters. Any of these promotersmay be cloned into multicopy (2μ) or single copy (CEN) plasmids to givean additional level of control in expression level. The plasmids caninclude nutritional markers (such as URA3, ADE3, HIS1, and others) forselection in yeast and antibiotic resistance (such as AMP) forpropagation in bacteria. Plasmids for expression on K. lactis are known,such as pKLAC1. Thus, in one example, after amplification in bacteria,plasmids can be introduced into the corresponding yeast auxotrophs bymethods similar to bacterial transformation.

The Mtb polypeptides can be expressed in a variety of yeast strains. Forexample, seven pleiotropic drug-resistant transporters, YOR1, SNQ2,PDR5, YCF1, PDR10, PDR11, and PDR15, together with their activatingtranscription factors, PDR1 and PDR3, have been simultaneously deletedin yeast host cells, rendering the resultant strain sensitive to drugs.Yeast strains with altered lipid composition of the plasma membrane,such as the erg6 mutant defective in ergosterol biosynthesis, can alsobe utilized. Proteins that are highly sensitive to proteolysis can beexpressed in a yeast strain lacking the master vacuolar endopeptidasePep4, which controls the activation of other vacuolar hydrolases.Heterologous expression in strains carrying temperature-sensitive (ts)alleles of genes can be employed if the corresponding null mutant isinviable.

Viral vectors encoding the Mtb polypeptides disclosed herein can also beprepared. A number of viral vectors have been constructed, includingpolyoma, SV40 (Madzak et al., 1992, J. Gen. Virol. 73:15331536),adenovirus (Berkner, 1992, Curr. Top. Microbiol. Immunol. 158:39-6;Berliner et al., 1988, BioTechniques 6:616-629; Gorziglia et al., 1992,J. Virol. 66:4407-4412; Quantin et al., 1992, Proc. Natl. Acad. Sci. USA89:2581-2584; Rosenfeld et al., 1992, Cell 68:143-155; Wilkinson et al.,1992, Nucl. Acids Res. 20:2233-2239; Stratford-Perricaudet et al., 1990,Hum. Gene Ther. 1:241-256), vaccinia virus (Mackett et al., 1992,Biotechnology 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr.Top. Microbiol. Immunol. 158:91-123; On et al., 1990, Gene 89:279-282),herpes viruses including HSV and EBV (Margolskee, 1992, Curr. Top.Microbiol. Immunol. 158:67-90; Johnson et al., 1992, J. Virol.66:2952-2965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield etal., 1987, Mol. Neurobiol. 1:337-371; Fresse et al., 1990, Biochem.Pharmacol. 40:2189-2199), Sindbis viruses (Herweijer et al., 1995, Hum.Gene Ther. 6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879),alphaviruses (Schlesinger, 1993, Trends Biotechnol. 11:18-22; Frolov etal., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377) and retrovirusesof avian (Brandyopadhyay et al., 1984, Mol. Cell Biol. 4:749-754;Petropouplos et al., 1992, J. Virol. 66:3391-3397), murine (Miller,1992, Curr. Top. Microbiol. Immunol. 158:1-24; Miller et al., 1985, Mol.Cell Biol. 5:431-437; Sorge et al., 1984, Mol. Cell Biol. 4:1730-1737;Mann et al., 1985, J. Virol. 54:401-407), and human origin (Page et al.,1990, J. Virol. 64:5370-5276; Buchschalcher et al., 1992, J. Virol.66:2731-2739). Baculovirus (Autographa californica multinuclearpolyhedrosis virus; AcMNPV) vectors are also known in the art, and maybe obtained from commercial sources (such as PharMingen, San Diego,Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla,Calif.).

Thus, in one embodiment, the polynucleotide encoding an Mtb polypeptideis included in a viral vector. Suitable vectors include retrovirusvectors, orthopox vectors, avipox vectors, fowlpox vectors, capripoxvectors, suipox vectors, adenoviral vectors, herpes virus vectors, alphavirus vectors, baculovirus vectors, Sindbis virus vectors, vacciniavirus vectors and poliovirus vectors. Specific exemplary vectors arepoxvirus vectors such as vaccinia virus, fowlpox virus and a highlyattenuated vaccinia virus (MVA), adenovirus, baculovirus and the like.

Pox viruses useful in practicing the present methods include orthopox,suipox, avipox, and capripox virus. Orthopox include vaccinia,ectromelia, and raccoon pox. One example of an orthopox of use isvaccinia. Avipox includes fowlpox, canary pox and pigeon pox. Capripoxinclude goatpox and sheep pox. In one example, the suipox is swinepox.Examples of pox viral vectors for expression as described for example,in U.S. Pat. No. 6,165,460, which is incorporated herein by reference.Other viral vectors that can be used include other DNA viruses such asherpes virus and adenoviruses, and RNA viruses such as retroviruses andpoliovirus.

In some cases, vaccinia viral vectors may elicit a strong antibodyresponse. Thus, while numerous boosts with vaccinia vectors arepossible, its repeated use may not be useful in certain instances.However, this sensitivity problem can be minimized by using pox fromdifferent genera for boosts. In one example, when the first or initialpox virus vector is vaccinia, the second and subsequent pox virusvectors are selected from the pox viruses from a different genus such assuipox, avipox, capripox or an orthopox immunogenically distinct fromvaccinia.

The vaccinia virus genome is known in the art. It is composed of a HINDF13L region, TK region, and an HA region. Recombinant vaccinia virus hasbeen used to incorporate an exogenous gene for expression of theexogenous gene product (see, for example, Perkus et al. Science229:981-984, 1985; Kaufman et al. Int. J. Cancer 48:900-907, 1991; Moss,Science 252:1662, 1991). A gene encoding an antigen of interest, such asan immunogenic Mtb polypeptide, can be incorporated into the HIND F13Lregion or alternatively incorporated into the TK region of recombinantvaccinia virus vector (or other nonessential regions of the vacciniavirus genome). Baxby and Paoletti (Vaccine 10:8-9, 1992) disclose theconstruction and use as a vector, of the non-replicating poxvirus,including canarypox virus, fowlpox virus and other avian species. Sutterand Moss (Proc. Natl. Acad. Sci. U.S.A. 89:10847-10851, 1992) and Sutteret al. (Vaccine 12:1032-1040, 1994) disclose the construction and use asa vector of the non-replicating recombinant Ankara virus (MVA, modifiedvaccinia Ankara).

Suitable vectors are disclosed, for example, in U.S. Pat. No. 6,998,252,which is incorporated herein by reference. In one example, a recombinantpoxvirus, such as a recombinant vaccinia virus is synthetically modifiedby insertion of a chimeric gene containing vaccinia regulatory sequencesor DNA sequences functionally equivalent thereto flanking DNA sequenceswhich in nature are not contiguous with the flanking vaccinia regulatoryDNA sequences that encode a Mtb polypeptide. The recombinant viruscontaining such a chimeric gene is effective at expressing the Mtbpolypeptide. In one example, the vaccine viral vector comprises (A) asegment comprised of (i) a first DNA sequence encoding a Mtb polypeptideand (ii) a poxvirus promoter, wherein the poxvirus promoter is adjacentto and exerts transcriptional control over the DNA sequence encoding anMtb polypeptide; and, flanking said segment, (B) DNA from a nonessentialregion of a poxvirus genome. The viral vector can encode a selectablemarker. In one example, the poxvirus includes, for example, a thymidinekinase gene (see U.S. Pat. No. 6,998,252, which is incorporated hereinby reference).

Viral vectors, such as poxviral vectors, that encode an Mtb polypeptideinclude at least one expression control element operationally linked tothe nucleic acid sequence encoding the Mtb polypeptide. The expressioncontrol elements are inserted in the viral vector to control andregulate the expression of the nucleic acid sequence. Examples ofexpression control elements of use in these vectors includes, but is notlimited to, lac system, operator and promoter regions of phage lambda,yeast promoters and promoters derived from polyoma, adenovirus,retrovirus or SV40. Additional operational elements include, but are notlimited to, leader sequence, termination codons, polyadenylation signalsand any other sequences necessary for the appropriate transcription andsubsequent translation of the nucleic acid sequence encoding the Mtbpolypeptide in the host system. The expression vector can containadditional elements necessary for the transfer and subsequentreplication of the expression vector containing the nucleic acidsequence in the host system. Examples of such elements include, but arenot limited to, origins of replication and selectable markers. It willfurther be understood by one skilled in the art that such vectors areeasily constructed using conventional methods (Ausubel et al., (1987) in“Current Protocols in Molecular Biology,” John Wiley and Sons, New York,N.Y.) and are commercially available.

Basic techniques for preparing recombinant DNA viruses containing aheterologous DNA sequence encoding the one or more Mtb polypeptides areknown in the art. Such techniques involve, for example, homologousrecombination between the viral DNA sequences flanking the DNA sequencein a donor plasmid and homologous sequences present in the parentalvirus (Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79:7415-7419).In particular, recombinant viral vectors such as a poxviral vector canbe used in delivering the gene. The vector can be constructed forexample by steps known in the art, such as steps analogous to themethods for creating synthetic recombinants of the fowlpox virusdescribed in U.S. Pat. No. 5,093,258, incorporated herein by reference.Other techniques include using a unique restriction endonuclease sitethat is naturally present or artificially inserted in the parental viralvector to insert the heterologous DNA.

Generally, a DNA donor vector contains the following elements: (i) aprokaryotic origin of replication, so that the vector may be amplifiedin a prokaryotic host; (ii) a gene encoding a marker which allowsselection of prokaryotic host cells that contain the vector (e.g., agene encoding antibiotic resistance); (iii) at least one DNA sequenceencoding the one or more Mtb polypeptide located adjacent to atranscriptional promoter capable of directing the expression of thesequence; and (iv) DNA sequences homologous to the region of the parentvirus genome where the foreign gene(s) will be inserted, flanking theconstruct of element (iii). Methods for constructing donor plasmids forthe introduction of multiple foreign genes into pox virus are describedin PCT Publication No. WO 91/19803, incorporated herein by reference.

Generally, DNA fragments for construction of the donor vector, includingfragments containing transcriptional promoters and fragments containingsequences homologous to the region of the parent virus genome into whichforeign DNA sequences are to be inserted, can be obtained from genomicDNA or cloned DNA fragments. The donor plasmids can be mono-, di-, ormultivalent (e.g., can contain one or more inserted foreign DNAsequences). The donor vector can contain an additional gene that encodesa marker that will allow identification of recombinant virusescontaining inserted foreign DNA. Several types of marker genes can beused to permit the identification and isolation of recombinant viruses.These include genes that encode antibiotic or chemical resistance (e.g.,see Spyropoulos et al., 1988, J. Virol. 62:1046; Falkner and Moss, 1988,J. Virol. 62:1849; Franke et al., 1985, Mol. Cell. Biol. 5:1918), aswell as genes such as the E. coli lacZ gene, that permit identificationof recombinant viral plaques by colorimetric assay (Panicali et al.,1986, Gene 47:193-199).

The DNA gene sequence to be inserted into the virus can be placed into adonor plasmid, such as an E. coli or a Salmonella plasmid construct,into which DNA homologous to a section of DNA such as that of theinsertion site of the poxvirus where the DNA is to be inserted has beeninserted. Separately the DNA gene sequence to be inserted is ligated toa promoter. The promoter-gene linkage is positioned in the plasmidconstruct so that the promoter-gene linkage is flanked on both ends byDNA homologous to a DNA sequence flanking a region of pox DNA that isthe desired insertion region. With a parental pox viral vector, a poxpromoter is used. The resulting plasmid construct is then amplified bygrowth within E. coli bacteria and isolated. Next, the isolated plasmidcontaining the DNA gene sequence to be inserted is transfected into acell culture, for example chick embryo fibroblasts, along with theparental virus, for example poxvirus. Recombination between homologouspox DNA in the plasmid and the viral genome respectively results in arecombinant poxvirus modified by the presence of the promoter-geneconstruct in its genome, at a site that does not affect virus viability.

As noted above, the DNA sequence is inserted into a region (insertionregion) in the virus that does not affect virus viability of theresultant recombinant virus. One of skill in the art can readilyidentify such regions in a virus by, for example, randomly testingsegments of virus DNA for regions that allow recombinant formationwithout seriously affecting virus viability of the recombinant. Oneregion that can readily be used and is present in many viruses is thethymidine kinase (TK) gene. The TK gene has been found in all pox virusgenomes examined, including leporipoxvirus (Upton et al., 1986, J.Virol. 60:920); shope fibroma virus; capripoxvirus (Gershon et al.,1989, J. Gen. Virol. 70:525); Kenya sheep-1; orthopoxvirus (Weir et al.,1983, J. Virol. 46:530); vaccinia (Esposito et al., 1984, Virology135:561); monkeypox and variola virus (Hruby et al., 1983, Proc. Natl.Acad. Sci. USA 80:3411); vaccinia (Kilpatrick et al., 1985, Virology143:399); Yaba monkey tumor virus; avipoxvirus (Binns et al., 1988, J.Gen. Virol. 69:1275); fowlpox; (Boyle et al., 1987, Virology 156:355;Schnitzlein et al., 1988, J. Virol. Meth. 20:341); and entomopox (Lytvynet al., 1992, J. Gen. Virol. 73:3235-3240). In vaccinia, in addition tothe TK region, other insertion regions include, for example, the HindIIIM fragment. In fowlpox, in addition to the TK region, other insertionregions include, for example, the BamHI J fragment (Jenkins et al.,1991, AIDS Res. Hum. Retroviruses 7:991-998), the EcoRI-HindIIIfragment, EcoRV-HindIII fragment, BamHI fragment and the HindIIIfragment set forth in EPO Application No. 0 308220 A1 (see also Calvertet al., 1993, J. Virol. 67:3069-3076; Taylor et al., 1988, Vaccine6:497-503; Spehner et al., 1990, J. Virol. 64:527-533; Boursnell et al.,1990, J. Gen. Virol. 71:621-628).

In swinepox, insertion sites include the thymidine kinase gene region.In addition to the requirement that the gene be inserted into aninsertion region, successful expression of the inserted gene by themodified poxvirus requires the presence of a promoter operably linked tothe desired gene. Generally, the promoter is placed so that it islocated upstream from the gene to be expressed. Promoters are well knownin the art and can readily be selected depending on the host and thecell type to be targeted. In one example, in poxviruses, pox viralpromoters are used, such as the vaccinia 7.5K, 40K or fowlpox promoterssuch as FPV CIA. Enhancer elements can also be used in combination toincrease the level of expression. Furthermore, inducible promoters canbe utilized.

Homologous recombination between donor plasmid DNA and viral DNA in aninfected cell can result in the formation of recombinant viruses thatincorporate the desired elements. Appropriate host cells for in vivorecombination are generally eukaryotic cells that can be infected by thevirus and transfected by the plasmid vector. Examples of such cellssuitable for use with a pox virus are chick embryo fibroblasts, HuTK143(human) cells, and CV-1 and BSC-40 (both monkey kidney) cells. Infectionof cells with pox virus and transfection of these cells with plasmidvectors is accomplished by techniques standard in the art (see U.S. Pat.No. 4,603,112 and PCT Publication No. WO 89/03429).

Following in vivo recombination, recombinant viral progeny can beidentified by one of several techniques. For example, if the DNA donorvector is designed to insert foreign genes into the parent virusthymidine kinase (TK) gene, viruses containing integrated DNA will beTK⁻ and can be selected on this basis (Mackett et al., 1982, Proc. Natl.Acad. Sci. USA 79:7415). Alternatively, co-integration of a geneencoding a marker or indicator gene with the foreign gene(s) ofinterest, as described above, can be used to identify recombinantprogeny. One specific non-limiting example of an indicator gene is theE. coli lacZ gene. Recombinant viruses expressing beta-galactosidase canbe selected using a chromogenic substrate for the enzyme (Panicali etal., 1986, Gene 47:193). Once a recombinant virus has been identified, avariety of well-known methods can be used to assay the expression of theMtb sequence encoded by the inserted DNA fragment. These methods includeblack plaque assay (an in situ enzyme immunoassay performed on viralplaques), Western blot analysis, radioimmunoprecipitation (RIPA), andenzyme immunoassay (EIA).

This disclosure encompasses a recombinant virus comprising more than oneantigen of interest for the purpose of having a multivalent vaccine. Forexample, the recombinant virus may comprise the virus genome or portionsthereof, the nucleic acid sequence encoding the Mtb polypeptide and anucleic acid sequence encoding a hepatitis B surface antigen or anyother carrier protein.

In one embodiment, a composition is provided that includes a recombinantvirus comprising a vaccinia virus genome or portions thereof, thenucleic acid sequence encoding an Mtb polypeptide and a recombinantvirus comprising the nucleic acid sequence encoding theimmunostimulatory molecule, B7-1 alone or in combination with thenucleic acid sequence encoding the immunostimulatory molecule, B7-2, ora recombinant virus containing both the genes for an Mtb polypeptide andan immunostimulatory molecule. This disclosure also encompasses arecombinant virus comprising the Mtb polypeptide that is administeredwith a second recombinant virus comprising the virus genome or portionthereof, and one or more nucleic acid sequences encoding one or more B7molecules, such as a recombinant vaccinia virus expressing B7-1 and/orB7-2.

Thus, in one example, recombinant virus is disclosed that is arecombinant vaccinia virus containing B7-1 and a recombinant vacciniavirus containing B7-2 (designated rV-B7-1 and rV-B7-2, respectively);the composition can include rV-B7-1 and/or rV-B7-2 in combination withan immunogenic Mtb polypeptide.

The B7 molecule includes but is not limited to B7-1, B7-2 and analogsthereof. The B7 gene may be cloned from mammalian sources, including butnot limited to mammalian tissues, genomic libraries or cDNA libraries,such as from murine or human sources. Without being bound by theory,costimulatory molecules of the B7 family (namely B7-1, B7-2, andpossibly B7-3) are believed to be members of the immunoglobulin genesuperfamily. These molecules are present on macrophages, dendriticcells, monocytes (antigen presenting cells (APCs)). Significantamplification of the immune response against a given antigen generallydoes not occur without co-stimulation (June et al., Immunology Today15:321-331, 1994; Chen et al., Immunology Today 14:483-486; Townsend etal., Science 259:368-370). Freeman et al. (J. Immunol. 143:2714-2722,1989) report cloning and sequencing of a B7-1 gene. Azuma et al. (Nature366:76-79, 1993) report cloning and sequencing of a B7-2 gene. Thus, inone embodiment the B7-1 gene or the B7-2 genes are administered inconjunction with the Mtb polypeptide. The insertion of nucleic acidsencoding B7-1 and B7-2 into vaccinia virus has been disclosed (see forexample, U.S. Pat. No. 6,893,869, incorporated herein by reference; thisU.S. Patent also discloses the use of a nucleic acid encoding IL-2 in avaccinia virus). Several vectors including IL-2, B7-1 and B7-2 have beendeposited with the American Type Culture Collection (ATCC) on Oct. 3,1994 under the terms of the Budapest Treaty (for example,rV-CEA/_(n)IL-2 (ATCC Designation VR 2480), rV-_(m)B7-2 (ATCCDesignation VR 2482); and rV-_(m)B7-1 (ATCC Designation VR 2483)).

DNA sequences encoding an Mtb polypeptide can be expressed in vitro byDNA transfer into a suitable host cell. The cell may be prokaryotic oreukaryotic. The term also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell since there may be mutations that occur during replication. Methodsof stable transfer, meaning that the foreign DNA is continuouslymaintained in the host, are known in the art.

As noted above, a polynucleotide sequence encoding an Mtb polypeptidecan be operatively linked to expression control sequences. An expressioncontrol sequence operatively linked to a coding sequence is ligated suchthat expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. The expression controlsequences include, but are not limited to, appropriate promoters,enhancers, transcription terminators, a start codon (i.e., ATG) in frontof a protein-encoding gene, splicing signal for introns, maintenance ofthe correct reading frame of that gene to permit proper translation ofmRNA, and stop codons.

Host cells can include microbial, yeast, insect and mammalian hostcells. Methods of expressing DNA sequences having eukaryotic or viralsequences in prokaryotes are well known in the art. Non-limitingexamples of suitable host cells include bacteria, archea, insect, fungi(for example, yeast), mycobacterium (such as M. smegmatis), plant, andanimal cells (for example, mammalian cells, such as human) Exemplarycells of use include E. coli, Bacillus subtilis, Saccharomycescerevisiae, Salmonella typhimurium, SF9 cells, C₁₂₉ cells, 293 cells,Neurospora, and immortalized mammalian myeloid and lymphoid cell lines.Techniques for the propagation of mammalian cells in culture arewell-known (see, Jakoby and Pastan (eds), 1979, Cell Culture. Meth.Enzymol., volume 58, Academic Press, Inc., Harcourt Brace Jovanovich,N.Y.). Examples of commonly used mammalian host cell lines are VERO andHeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although othercell lines may be used, such as cells designed to provide higherexpression, desirable glycosylation patterns, or other features. Asdiscussed above, techniques for the transformation of yeast cells, suchas polyethylene glycol transformation, protoplast transformation andgene guns are also known in the art (see Gietz and Woods, Meth. Enzymol.350: 87-96, 2002).

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as, but not limited to, E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method using procedures well known in the art.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding an Mtbpolypeptide, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

In particular embodiments provided herein, one or more of the disclosedMtb polynucleotides (or fragments thereof) can be conjugated to asubstrate or solid support, such as a plate or array. In one example,the plate or array includes, consists essentially of, or consists of one(such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all)of SEQ ID NOs: 19-36 or fragments thereof. In some examples, the plateor array also includes one or more control polynucleotides. Methods forselecting an appropriate substrate and constructing a plate or array arewell known to one of skill in the art (see, e.g., U.S. Pat. Nos.5,554,501; 5,985,567; 5,981,185; and 6,013,789; and PCT Publications WO85/01051 and WO 89/10977; all incorporated herein by reference).

IV. THERAPEUTIC METHODS AND PHARMACEUTICAL COMPOSITIONS

The Mtb polypeptides disclosed herein, or nucleic acids encoding the Mtbpolypeptides, can be used to generate an immune response in a subject.In several examples, the subject is infected with Mtb or is at risk ofbeing infected with Mtb. Thus, in several embodiments, the methodsinclude administering to a subject a therapeutically effective amount ofone or more of the Mtb polypeptides disclosed herein (or polynucleotidesencoding these polypeptides) in order to generate an immune response,such as, but not limited to, a protective immune response.

In exemplary applications, compositions are administered to a subject inan amount sufficient to produce an immune response to Mtb. These Mtbpolypeptides, or polynucleotides encoding these polypeptides, are of useto inhibit or prevent an infection with Mtb, inhibit or preventprogression to disease in a subject having a latent Mtb infection, or totreat tuberculosis in a subject infected with Mtb. In several examples,administration of a therapeutically effective amount of a compositionincluding one or more of the Mtb polypeptides disclosed herein (orpolynucleotides encoding these polypeptides) induces a sufficient immuneresponse to decrease a symptom of a disease due to Mtb infection, toinhibit the development of one or more symptoms of tuberculosis, or toinhibit infection with Mtb.

In some examples, the compositions are of use in inhibiting orpreventing a future infection with Mtb. Thus, a therapeuticallyeffective amount of the composition is administered to a subject at riskof becoming infected with Mtb. The composition inhibits or prevents thedevelopment of tuberculosis, such as latent or active tuberculosis, inthe subject upon subsequent exposure to Mtb.

In additional examples, the compositions are administered to a subjectwith a latent Mtb infection, and inhibit or prevent the development ofsymptoms of tuberculosis. In some examples, the compositions are of usein treating a subject with latent tuberculosis, such that the subjectdoes not develop active tuberculosis.

Amounts effective for these uses will depend upon the severity of thedisease, the general state of the patient's health, and the robustnessof the patient's immune system. In one example, a therapeuticallyeffective amount of the compound is that which provides eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Inother examples, a therapeutically effective amount is an amountsufficient to inhibit an infection with Mtb in a subject upon subsequentexposure of the subject to Mtb. In additional examples, atherapeutically effective amount is an amount sufficient to inhibitdevelopment of one or more symptoms in a subject infected with Mtb.

In some examples, one or more Mtb polypeptide described herein may becovalently linked to at least one other immunogenic protein, wherein theconjugate elicits an immune response to the Mtb polypeptide in asubject. The other immunogenic protein (sometimes referred to as a“carrier” protein) ideally has the properties of being immunogenic byitself, usable in a subject, and of a size that can be easily purifiedand conjugated to at least one other protein or peptide. Suitablecarrier proteins are known to one of skill in the art. In particularexamples, the other immunogenic protein (carrier protein) is bovineserum albumin (BSA), ovalbumin, tetanus toxoid, diphtheria toxoid,cholera toxin, Clostridium difficile toxin A, C. difficile toxin B,Shiga toxin, or Pseudomonas aeruginosa recombinant exoprotein A.

An Mtb polypeptide can be administered by any means known to one ofskill in the art (see Banga, A., “Parenteral Controlled Delivery ofTherapeutic Peptides and Proteins,” in Therapeutic Peptides andProteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995) eitherlocally or systemically, such as by intramuscular injection,subcutaneous injection, intraperitoneal injection, intravenousinjection, oral administration, nasal administration, transdermaladministration, or even anal administration. In one embodiment,administration is by oral administration, subcutaneous injection, orintramuscular injection. To extend the time during which the peptide orprotein is available to stimulate a response, the peptide or protein canbe provided as an implant, an oily injection, or as a particulatesystem. The particulate system can be a microparticle, a microcapsule, amicrosphere, a nanocapsule, or similar particle. (see, e.g., Banga,supra). A particulate carrier based on a synthetic polymer has beenshown to act as an adjuvant to enhance the immune response, in additionto providing a controlled release. Aluminum salts can also be used asadjuvants to produce an immune response.

In one specific, non-limiting example, the Mtb polypeptide isadministered in a manner to direct the immune response to a cellularresponse (that is, a cytotoxic T lymphocyte (CTL) response), rather thana humoral (antibody) response.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF; one or more molecules such as OX-40L or 4-1 BBL, or combinationsof these molecules, can be used as biological adjuvants (see, forexample, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze etal., 2000, Cancer J. Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, StemCells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol.465:381-90). These molecules can be administered systemically (orlocally) to the host. In several examples, IL-2, RANTES, GM-CSF, TNF-α,IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1 B7-2, OX-40L, 4-1 BBL,and/or ICAM-1 are administered.

A number of means for inducing cellular responses, both in vitro and invivo, are known. Lipids have been identified as agents capable ofassisting in priming CTL in vivo against various antigens. For example,as described in U.S. Pat. No. 5,662,907, palmitic acid residues can beattached to the alpha and epsilon amino groups of a lysine residue andthen linked (for example, via one or more linking residues, such asglycine, glycine-glycine, serine, serine-serine, or the like) to animmunogenic peptide. The lipidated peptide can then be injected directlyin a micellar form, incorporated in a liposome, or emulsified in anadjuvant. As another example, E. coli lipoproteins, such astripalmitoyl-S-glycerylcysteinlyseryl-serine can be used to prime tumorspecific CTL when covalently attached to an appropriate peptide (see,Deres et al., Nature 342:561, 1989). Further, as the induction ofneutralizing antibodies can also be primed with the same moleculeconjugated to a peptide which displays an appropriate epitope, twocompositions can be combined to elicit both humoral and cell-mediatedresponses where that is deemed desirable.

A pharmaceutical composition including an Mtb polypeptide is thusprovided. These compositions are of use to promote an immune response toMtb. In one embodiment, the Mtb polypeptide is mixed with an adjuvantcontaining two or more of a stabilizing detergent, a micelle-formingagent, and an oil. Suitable stabilizing detergents, micelle-formingagents, and oils are detailed in U.S. Pat. No. 5,585,103; U.S. Pat. No.5,709,860; U.S. Pat. No. 5,270,202; and U.S. Pat. No. 5,695,770, all ofwhich are incorporated by reference. A stabilizing detergent is anydetergent that allows the components of the emulsion to remain as astable emulsion. Such detergents include polysorbate, 80 (TWEEN)(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured byICI Americas, Wilmington, Del.), TWEEN 40™, TWEEN 20™, TWEEN 60™,ZWITTERGENT™ 3-12, TEEPOL HB7™, and SPAN 85™. These detergents areusually provided in an amount of approximately 0.05 to 0.5%, such as atabout 0.2%. A micelle forming agent is an agent which is able tostabilize the emulsion formed with the other components such that amicelle-like structure is formed. Such agents generally cause someirritation at the site of injection in order to recruit macrophages toenhance the cellular response. Examples of such agents include polymersurfactants described by BASF Wyandotte publications, e.g., Schmolka, J.Am. Oil. Chem. Soc. 54:110, 1977; and Hunter et al., J. Immunol.127:1244-1250, 1981; for example, PLURONIC™ L62LF, L101, and L64,PEG1000, and TETRONIC™ 1501, 150R1, 701, 901, 1301, and 130R1. Thechemical structures of such agents are well known in the art. In oneembodiment, the agent is chosen to have a hydrophile-lipophile balance(HLB) of between 0 and 2, as defined by Hunter and Bennett, J. Immun.133:3167, 1984. The agent can be provided in an effective amount, forexample between 0.5 and 10%, or in an amount between 1.25 and 5%.

The oil included in the composition is chosen to promote the retentionof the antigen in oil-in-water emulsion, such as to provide a vehiclefor the desired antigen, and preferably has a melting temperature ofless than 65° C. such that emulsion is formed either at room temperature(about 20° C. to 25° C.), or once the temperature of the emulsion isbrought down to room temperature. Examples of such oils includesqualene, squalane, EICOSANE™, tetratetracontane, glycerol, and peanutoil or other vegetable oils. In one specific, non-limiting example, theoil is provided in an amount between 1 and 10%, or between 2.5 and 5%.The oil should be both biodegradable and biocompatible so that the bodycan break down the oil over time, and so that no adverse affects, suchas granulomas, are evident upon use of the oil.

In one embodiment, the adjuvant is a mixture of stabilizing detergents,micelle-forming agent, and oil available under the name PROVAX® (IDECPharmaceuticals, San Diego, Calif.). An adjuvant can also be animmunostimulatory nucleic acid, such as a nucleic acid including a CpGmotif, or a biological adjuvant (see above).

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems, see Banga, Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., 1995. Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein as acentral core. In microspheres, the therapeutic agent is dispersedthroughout the particle. Particles, microspheres, and microcapsulessmaller than about 1 μm are generally referred to as nanoparticles,nanospheres, and nanocapsules, respectively. Capillaries have a diameterof approximately 5 μm so that only nanoparticles are administeredintravenously. Microparticles are typically around 100 μm in diameterand are administered subcutaneously or intramuscularly (see Kreuter,Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc.,New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on ControlledDrug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y.,pp. 315-339, 1992).

Polymers can be used for controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., InL J. Pharm. 112:215,1994). In yet another aspect, liposomes are used for controlled releaseas well as drug targeting of the lipid-capsulated drug (Betageri et al.,Liposome Drug Delivery Systems, Technomic Publishing Co., Inc.,Lancaster, Pa., 1993). Numerous additional systems for controlleddelivery of therapeutic proteins are known (e.g., U.S. Pat. Nos.5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735;5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697;4,902,505; 5,506,206; 5,271,961; 5,254,342; and 5,534,496).

In another embodiment, a pharmaceutical composition includes a nucleicacid encoding an Mtb polypeptide. A therapeutically effective amount ofthe Mtb polynucleotide can be administered to a subject in order togenerate an immune response.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF, one or more costimulatory molecules, such as ICAM-1, LFA-3, CD72,B7-1, B7-2, or other B7 related molecules; one or more molecules such asOX-40L or 4-1 BBL, or combinations of these molecules, can be used asbiological adjuvants (see, for example, Salgaller et al., 1998, J. Surg.Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J. Sci. Am. 6(Suppl1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper etal., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can beadministered systemically to the host. It should be noted that thesemolecules can be co-administered via insertion of a nucleic acidencoding the molecules into a vector, for example, a recombinant poxvector (see, for example, U.S. Pat. No. 6,045,802). In variousembodiments, the nucleic acid encoding the biological adjuvant can becloned into same vector as the Mtb polypeptide coding sequence, or thenucleic acid can be cloned into one or more separate vectors forco-administration. In addition, nonspecific immunomodulating factorssuch as BCG and levamisole can be co-administered.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Asdescribed above, the nucleotide sequence encoding an Mtb polypeptide canbe placed under the control of a promoter to increase expression of themolecule.

Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response), and U.S. Pat. Nos.5,593,972 and 5,817,637 (which describe operably linking a nucleic acidsequence encoding an antigen to regulatory sequences enablingexpression). U.S. Pat. No. 5,880,103 describes several methods ofdelivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™ (negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and saponin). Protective immunity has been generated in avariety of experimental models of infection, including toxoplasmosis andEpstein-Barr virus-induced tumors, using ISCOMS™ as the delivery vehiclefor antigens (Mowat and Donachie, Immunol. Today 12:383, 1991). Doses ofantigen as low as 1 μg encapsulated in ISCOMS™ have been found toproduce Class I mediated CTL responses (Takahashi et al., Nature344:873, 1990).

In another approach to using nucleic acids for immunization, an Mtbpolypeptide can also be expressed by attenuated viral hosts or vectorsor bacterial vectors. Recombinant vaccinia virus, adeno-associated virus(AAV), herpes virus, retrovirus, or other viral vectors can be used toexpress the peptide or protein, thereby eliciting a CTL response. Forexample, vaccinia vectors and methods useful in immunization protocolsare described in U.S. Pat. No. 4,722,848. BCG provides another vectorfor expression of the peptides (see Stover, Nature 351:456-460, 1991).

When a viral vector is utilized, it is desirable to provide therecipient with a dosage of each recombinant virus in the composition inthe range of from about 10⁵ to about 10¹⁰ plaque forming units, althougha lower or higher dose can be administered. The composition ofrecombinant viral vectors can be introduced into a mammal (1) prior toany evidence of an infection with Mtb; (2) to inhibit development oftuberculosis in an individual infected with Mtb; or (3) to decrease asymptom of tuberculosis in a mammal infected with Mtb. Examples ofmethods for administering the composition into mammals include, but arenot limited to, exposure of cells to the recombinant virus ex vivo, orinjection of the composition into the affected tissue or intravenous,subcutaneous, intradermal or intramuscular administration of the virus.Alternatively the recombinant viral vector or combination of recombinantviral vectors may be administered locally in a pharmaceuticallyacceptable carrier. Generally, the quantity of recombinant viral vector,carrying the nucleic acid sequence of one or more Mtb polypeptides to beadministered is based on the titer of virus particles. An exemplaryrange of the immunogen to be administered is 10⁵ to 10¹⁰ virus particlesper mammal, such as a human.

In the embodiment where a combination of a first recombinant viralvector carrying a nucleic acid sequence of one or more Mtb polypeptideand a second recombinant viral vector carrying the nucleic acid sequenceof one or more immunostimulatory molecules is used, the mammal can beimmunized with different ratios of the first and second recombinantviral vector. In one embodiment the ratio of the first vector to thesecond vector is about 1:1, or about 1:3, or about 1:5. Optimal ratiosof the first vector to the second vector may easily be titered using themethods known in the art (see, for example, U.S. Pat. No. 6,893,869,incorporated herein by reference).

In one embodiment the recombinant viruses have been constructed toexpress cytokines (such as TNF-α, IL-6, GM-CSF, and IL-2), andcostimulatory and accessory molecules (B7-1, B7-2) alone and in avariety of combinations. Simultaneous production of an immunostimulatorymolecule and the Mtb polypeptide enhances the generation of specificeffectors. Without being bound by theory, dependent upon the specificimmunostimulatory molecules, different mechanisms might be responsiblefor the enhanced immunogenicity: augmentation of help signal (IL-2),recruitment of professional APC (GM-CSF), increase in CTL frequency(IL-2), effect on antigen processing pathway and MHC expression (IFNγand TNFα) and the like. For example, IL-2, IL-6, interferon, tumornecrosis factor, or a nucleic acid encoding these molecules, can beadministered in conjunction with an Mtb immunogenic polypeptide, or anucleic acid encoding an Mtb polypeptide. The co-expression of an Mtbpolypeptide together with at least one immunostimulatory molecule can beeffective in an animal model to show therapeutic effects.

In one embodiment, a nucleic acid encoding an Mtb polypeptide isintroduced directly into cells. For example, the nucleic acid can beloaded onto gold microspheres by standard methods and introduced intothe skin by a device such as the Helios™ Gene Gun (Bio-Rad, Hercules,Calif.). The nucleic acids can be “naked,” consisting of plasmids undercontrol of a strong promoter. Typically, the DNA is injected intomuscle, although it can also be injected directly into other sites.Dosages for injection are usually around 0.5 μg/kg to about 50 mg/kg,and typically are about 0.005 mg/kg to about 5 mg/kg (see, for example,U.S. Pat. No. 5,589,466).

In one specific, non-limiting example, a pharmaceutical composition forintravenous administration would include about 0.1 μg to 10 mg ofimmunogenic Mtb polypeptide per patient per day. Dosages from 0.1 toabout 100 mg per patient per day can be used, particularly if the agentis administered to a secluded site and not into the circulatory or lymphsystem, such as into a body cavity or into a lumen of an organ. Actualmethods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remington's Pharmaceutical Sciences, 19^(th) Ed.,Mack Publishing Company, Easton, Pa., 1995.

Single or multiple administrations of the compositions are administered,depending on the dosage and frequency as required and tolerated by thesubject. In one embodiment, the dosage is administered once as a bolus,but in another embodiment can be applied periodically until atherapeutic result is achieved. In one embodiment, the dose issufficient to treat or ameliorate symptoms or signs of tuberculosiswithout producing unacceptable toxicity to the subject. In anotherembodiment, the dose is sufficient to inhibit infection with Mtb uponsubsequent exposure to Mtb. In a further embodiment, the dose issufficient to inhibit a symptom of tuberculosis in a subject with alatent Mtb infection. Systemic or local administration can be utilized.

In another method, antigen presenting cells (APCs), such as dendriticcells (DCs), are isolated from a subject of interest and pulsed orco-incubated with peptides comprising an Mtb polypeptide in vitro. Inone specific, non-limiting example, the APCs are autologous cellsisolated from the subject of interest. A therapeutically effectiveamount of the antigen presenting cells is administered (re-introduced)to the subject of interest.

The Mtb polypeptide can be delivered to the DCs or to DC precursors viaany method known in the art, including, but not limited to, pulsing DCsdirectly with antigen, or utilizing a broad variety of antigen deliveryvehicles, such as, for example, liposomes, or other vectors known todeliver antigen to cells. In one specific, non-limiting example anantigenic formulation includes about 0.1 μg to about 1,000 μg, or about1 to about 100 μg of a selected Mtb polypeptide. The Mtb polypeptide canalso be administered with agents that promote DC maturation. Specific,non-limiting examples of agents of use are interleukin-4 (IL-4) andgranulocyte/macrophage colony stimulating factor (GM-CSF), or flt-3ligand (fit-3L). The preparation can also contain buffers, excipients,and preservatives, amongst other ingredients.

In one embodiment, mature APCs are generated to present the immunogenicMtb polypeptide. These DCs are then administered alone to a subjectinfected with Mtb, or at risk for infection with Mtb. In anotherembodiment, the mature DCs are administered in conjunction with anantibacterial or antiviral agent.

Alternatively, the APCs are used to sensitize CD8 cells, such asperipheral blood lymphocytes (PBLs). The PBLs can be from the samesubject (autologous) that is to be treated. Alternatively, the PBLs canbe heterologous. However, they should at least be MHC Class-I restrictedto the HLA types the subject possesses. An effective amount of thesensitized cells is then administered to the subject.

Peripheral blood mononuclear cells (PBMCs) can be used as the respondercell source of cytotoxic T lymphocyte (CTL) precursors. The appropriateantigen-presenting cells are incubated with peptide, after which thepeptide-loaded antigen-presenting cells are then incubated with theresponder cell population under optimized culture conditions. PositiveCTL activation can be determined by assaying the culture for thepresence of CTLs that kill radio-labeled target cells, both specificpeptide-pulsed targets as well as target cells expressing endogenouslyprocessed forms of the antigen from which the peptide sequence wasderived.

Alternatively, a CD8⁺ T cell clone that recognizes the Mtb polypeptidecan be isolated from a subject of interest. This CD8⁺ T cell clone canbe expanded in vitro, using methods known in the art. A therapeuticallyeffective amount of the CD8⁺ T cells is then administered to the subjectof interest.

Thus, cells can be administered to a subject to treat, inhibit, or evenprevent an Mtb infection, such as to decrease a symptom of an Mtbinfection. In these applications, a therapeutically effective amount ofactivated APCs, or activated lymphocytes, are administered to a subjectin an amount sufficient to raise an immune response to Mtb.

In supplemental methods, any therapeutic regimen is augmented byadministering a cytokine, such as interleukin (IL)-2, IL-3, IL-6, IL-10,IL-12, IL-15, GM-CSF, interferons. In further methods, an additionalantibacterial or antiviral agent is administered to the subject.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Selection of Antigens

A peptide library encompassing 39,499 Mtb peptides was screened forantigens and/or epitopes that were both strongly and commonly recognizedin individuals with Mtb infection in Portland, Oreg. This peptidelibrary represents 389 genes, representing roughly 10% of the Mtbgenome. The peptides are 15 mers overlapping by 11 amino acids for eachgene product. 50 nmol of each peptide was synthesized individually andthen pooled into 777 pools of 50 peptides in a 96 well format (nineplates). Five blank wells and one well of an irrelevant peptide pool,SIV gag, were included on each of the nine plates.

CD8⁺ T cells from donors were screened against the peptide library byIFN-γ ELISPOT. The IFN-γ ELISPOT assay was performed as describedpreviously (Beckman et al., J. Immunol. 157:2795-2803, 1996). Fordetermination of ex vivo frequencies of CD8⁺ T cells responding to Mtbinfection or Mtb antigens, CD8⁺ T-cells were positively selected fromperipheral blood mononuclear cells using magnetic beads (MiltenyiBiotec, Auburn Calif.) as a source of responder T cells and tested fortheir response to autologous DC. Each plate of the genomic peptidelibrary was screened in duplicate, for a total of 18 ELISPOT plates perscreen. CD8+ T cells were prepared from cryopreserved PBMC by CD8selection using magnetic bead separations. Resulting cell populationscontained >99% CD8+ T cells. CD8+ T cells (250,000 cells/well),autologous DCs (20,000 cells/well), and IL-2 (0.5 ng/ml) were added topeptide (final 5 ug/ml, individual peptides) in the ELISPOT plates. Fivemedia control wells were included on each plate. Spots are enumeratedusing with the AID EliSpot Reader System. For each plate, the mean ofthese five wells was subtracted from each well of that plate tonormalize between plates. Each technical replicate on each plate wasthen scored. A well was scored positive if the spot forming units (SFU),less the mean of the media wells, was greater than or equal to ten andthe SFU was greater than or equal to twice the mean of the media. Twentydonors were tested, including fifteen LTBI (6 Caucasian, 4 AfricanAmerican, 5 SE Asian) and five donors with active TB.

Two criteria were used to select the peptide pools. First, peptide poolshad to be in the top 5% of a donor's response. Second, the peptide poolhad to be recognized by three or more donors. The peptide pools selectedby this method were identical independent of the order these criteriawere applied. A well was considered positive in the donor screen if onlyone technical replicate was statistically positive. However, since thereis more confidence in a well where both technical replicates arepositive, the selected wells were compared if the average spot formingunits (SFU) for wells with two positive technical replicates wasweighted by 200% to the selected wells if the average SFU was notweighted. 32 wells were selected if there was no weighting given to thetechnical replicates and 35 wells were selected if the weighting wasapplied. However, 19 wells were selected by both weighting and notweighting the average SFU and these were chosen for further analysis(Table 2).

TABLE 2 Selected antigens and epitopes for clinical validation studiesAntigen Number Rv Numbers Gene Names 1 Rv3641c (33)¹ fic 2 Rv3136 (46):Rv3135 (4) PPE51: PPE50 3 Rv0383c (30): Rv0394c (20) Rv0383c: Rv0394c 4Rv1184c (20) Rv1184c 5 Rv3514 (47): Rv3532 (3) PE_PGRS57: PPE61 6 Rv3558(44): Rv3539 (6) PPE64: PPE63 7 Rv1979c (50) Rv1979c 8 Rv1980c (28):Rv1984c (22) mpt64: cfp21 9 Rv3347c (50) PPE55 10 Rv0151c (50) PE1 11Rv1997 (50) ctpF 12 Rv1997 (50) ctpF 13 Rv0159c (50) PE3 14 Rv1997 (50)ctpF 15 Rv2711 (37): Rv1404 (13) ideR: Rv1404 16 Rv1706c (50) PPE23 17Rv2041c (50) Rv2041c 18 Rv2041c (43): Rv2093c (7) Rv2041c: tatC 19Rv1039c (50) PPE15 ¹Number of peptides from each gene shown inparentheses

Example 2 Screening of Selected Antigens

The antigens identified in Example 1 were screened in a CD8 ELISPOTassay against latent and active TB donors from Uganda. ELISPOT plateswere read using the AID ELISPOT reader and output was exported intoexcel files. Data were imported into SAS® version 9.1 (SAS Institute,Inc., Cary, N.C.) and analyzed. A categorical variable for a positiveELISPOT was created in SAS®. For a positive response to the antigen, themean of the antigen containing wells must be greater than the backgroundwells by two standard deviations. If this was true, the background wassubtracted and this difference must then be greater than 10 spots.Similarly, a continuous ELISPOT variable was created for each antigendetailing the spot forming units remaining if the antigen met thecategorical criteria above. The results were graphed by proportion ofpositive responses stratified by active or latent TB along with thecorresponding spot forming unit (FIGS. 1A and B).

Five antigens were selected for the validation stage. Several factorswere considered in the selection, including those antigens that had asuggestion of disease specificity, as well as antigens with a broad andstrong response. These antigens included PPE50:51, PE3, CtpF, PPE15, andEsxJ. Fifty-six latent and 52 active TB individuals were studied in thevalidation phase. Twenty-one individuals (19.2%) responded to all fiveantigens at the predefined cut-off, whereas 10 individuals (9%)responded to four of the antigens. Forty individuals (36%) responded toup to three antigens and 35% did not respond to any of the five antigensselected. Although some disease specificity was noted in the screeningstage, especially as it applied to PPE50:51, this was not apparent inthe validation stage.

The magnitude of the response was studied as well. Using Poissonmodeling, individuals with latent disease had a significantly greaterspot count than those with active disease for 4 antigens (PPE50:51,cTPF, PPE15, EsXJ) however the difference was not clinically meaningful(FIG. 2).

Example 3 Additional Antigens

Additional antigens were selected using the methods described inExample 1. The additional antigens are provided in Table 3.

The additional identified antigens were screened in a CD8 ELISPOT assay(as described in Examples 1 and 2) against latent and active TB donorsfrom Uganda. The results were graphed by the corresponding spot formingunit (FIG. 3).

TABLE 3 Additional antigens and epitopes for clinical validation studiesRv_Numbers (# peptides in pool) Gene_Names Rv0284(17): Rv0288(11)Rv0284: esxH Rv0917(31) betP Rv1243c(50) PE_PGRS23 Rv3345c(100)PE_PGRS50 Rv3163c(41): Rv3194c(9) Rv3163c: Rv3194c Rv0977(50) PE_PGRS16Rv0152c(40): Rv0151c(10) PE2: PE1 Rv1917c(50) PPE34 Rv2040c(37):Rv2025c(13) Rv2040c: Rv2025c Rv2356c(50) PPE40 Rv3159c(50) PPE53Rv1172c(32): Rv1195(18) PE12: PE13 Rv1348(35): Rv1343c(15) Rv1348: 1prDRv3873(50) PPE68

Example 4 Identification of Peptide-Specific T Cell Clones

Peptide-specific T cell clones were isolated from individuals with LTBIor active TB, using peptide pulsed DCs as APCs and limiting dilutioncloning methodology. Briefly, CD8+ T cells were isolated from PBMCsusing positive selection using CD8 antibody-coated magnetic beads perthe manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach,Germany). T cells were seeded at various concentrations in the presenceof a 2×10⁴—irradiated autologous peptide pulsed DC, 1×10⁵ irradiatedautologous PBMC, and rIL-2 (5 ng/ml) in cell culture media consisting of200 μl of RPMI 1640 supplemented with 10% human sera. Wells exhibitinggrowth between 10-14 days were assessed for peptide specificity usingELISPOT and peptide pulsed DCs as a source of APCs. T cells retainingpeptide specificity were further phenotyped for αβ T cell receptorexpression and CD8 expression by FACS.

Using the 15 mer Rv3136₁₃₇₋₁₅₁, T cell clones were generated to thepeptide using the methods described. Having derived an antigen-specificCD8⁺ T cell clone, the minimal epitope was determined. The minimalepitope was defined as the epitope which allowed for T cell recognitionat the lowest concentration of peptide. Each 9-mer, 10-mer, and 11-merpeptide within the 15-mer was tested over a broad range of peptideconcentrations, and by definition, the peptide eliciting a response atthe lowest peptide concentration is the minimal epitope. Peptidesincluding amino acids 139-149 of Rv3136 (SEQ ID NO: 2) allowed for Tcell recognition at the lowest concentrations (FIG. 4), with amino acids141-49 eliciting a response at the lowest concentration of all testedpeptides.

Example 5 Animal Models

In tuberculosis research, mouse and guinea pig models have been usedextensively to model various aspects of the disease.

A. Mouse Model:

Mice can be infected by a variety of routes, including intravenous,intraperitoneal and tracheal. One route is aerosolization of theinfectious organism for respiratory infection. The mice are exposed tothe aerosol in a chamber (wither whole body or nose only infection). Thedose of invention can be varied by manipulating the concentration of Mtbin the nebulizer or time of exposure. A low dose infection, such asabout 50 colony forming units (CFU) via aerosol, results in a slow andsteady increase in bacterial numbers in the lungs, generally reaching apeak in four weeks, which coincides with the peak number of T cells inthe lungs. The initial period is considered the acute stage ofinfection. Following infection, there is a dissemination of bacteria tothe mediastinal lymph nodes. T cell priming is generally detectablebetween two and three weeks. After about four weeks the bacterialnumbers stabilize, and there is a slow progressive pathologic response.This system is of use for modeling active infection. Thus, theabove-described polypeptides, or polynucleotides encoding thesepolypeptides, can be administered prior to infection. The ability of theMtb polypeptides (or polynucleotides encoding these polypeptides) toinhibit or prevent infection is then assessed. Alternatively, the miceare administered Mtb, and the ability of the Mtb polypeptide (orpolynucleotide encoding these polypeptides) to treat the Mtb infectionis monitored. The effectiveness of the Mtb polypeptides (orpolynucleotides) can be monitored by measuring the T cell response, suchas the number of CD8⁺ or CD4⁺ T cells, and/or measuring the bacterialnumbers, and/or evaluating the pathology.

Exemplary protocols are provided below (see also Repique et al., Infec.Immun. 70: 3318-3323, 2002, incorporated herein by reference for anadditional protocol).

1. Short Term Mouse Model:

C₅₇BL/6 mice are vaccinated with a composition including one or more Mtbpolypeptide, or a polynucleotide encoding these one or more polypeptidesaccording to the appropriate protocol and then rested for 4 to 6 weeksImmunized mice are infected with a low dose aerosol (50-100 CFU) ofvirulent M. tuberculosis and protection is evaluated by assessing thenumber of viable bacilli 30 days post challenge.

Viable counts are performed on the lung and spleen of mice byhomogenizing the organs and plating serial 10-fold dilutions on 7H11agar plates. Plates are incubated for up to 21 days and the number ofcolony forming units per organ determined.

BCG vaccinated mice have approximately 1 Log₁₀ protection in their lungand spleen when compared to PBS-treated mice.

B. Guinea Pig Models:

1. Short Term Guinea Pig Model

Out-bred Hartley guinea pigs are vaccinated with a composition includingone or more Mtb polypeptide, or a polynucleotide encoding these one ormore polypeptides, and then rested for 8 to 10 weeks Immunized guineapigs are infected with a low dose aerosol (10-30 CFU) of virulent M.tuberculosis and protection is evaluated by assessing the number ofviable bacilli 30 days post challenge.

Viable counts are performed on the lung and spleen of guinea pigs byhomogenizing the organs and plating serial 10-fold dilutions on 7H11agar plates. Plates are incubated for up to 21 days and the number ofcolony forming units per organ determined. Lung and spleen segments arealso taken for histological analyses.

BCG vaccinated guinea pigs have approximately 2-3 Log₁₀ protection intheir lung and spleen when compared to PBS-treated guinea pigs. Inaddition, BCG vaccinated guinea pigs have well defined granulomas whencompared to unvaccinated animals.

2. Long Term Guinea Pig Model

The guinea pig model is similar to the mouse model, but the experimentsare open-ended survival type and can last for as long as 2 years. Guineapigs develop “classical” granulomas similar to humans with active TB,and as lung tissue necrosis progresses, they begin to lose weight anddie of TB similar to humans. The number of colony forming units in thelungs and spleen can be assessed. Histological examination can also beperformed to determine the degree of lung involvement and tissuedestruction. After low-dose aerosol exposure in the guinea pig thenumber of organisms increases progressively during the first three weeksand then plateaus into a chronic state. During the later stages ofinfection there is increased bacterial load in the lung and this isassociated with a worsening pathological condition. Without treatment,there is a concomitant rise in both CD4 and CD8 T cells in the lungs ofinfected guinea pigs.

Out-bred Hartley guinea pigs are vaccinated with the experimentalvaccine (such as a composition including one or more Mtb polypeptide, ora polynucleotide encoding these one or more polypeptides) according tothe appropriate protocol and then rested for 8 to 10 weeks Immunizedguinea pigs are then infected with a low dose aerosol (10-30 CFU) ofvirulent M. tuberculosis. Guinea pigs are weighed weekly and monitoreddaily for signs of disease (such as increased respiration and failure tothrive). Unvaccinated guinea pigs succumb to infection from 20 to 25weeks post challenge, while BCG vaccinated guinea pigs survive for 50 to55 weeks post challenge.

At necropsy, the lung and spleen are assessed for the number of CFU andthe extent of pathology. The relative protection of the experimentalcomposition is compared to BCG vaccinated animals.

Example 6 Methods of Treating or Inhibiting Tuberculosis in a Subject

This example describes methods that can be used to induce an immuneresponse in a subject that has or is at risk of having tuberculosis. Inparticular examples, the method includes selecting a subject having,thought to have, or at risk of having tuberculosis. Subjects having orthought to have tuberculosis include those with symptoms such aspersistent cough, blood-tinged sputum, fever, weight loss, Ghon complex,or a positive diagnostic test (such as a tuberculin skin test). Subjectsat risk of tuberculosis include those with exposure to an infectedindividual, those in an area where tuberculosis is endemic, andimmunocompromised individuals.

Subjects selected for treatment can be administered a therapeutic amountof a disclosed immunogenic Mtb polypeptide or immunogenic fragmentthereof or a polynucleotide encoding the polypeptide or fragmentthereof. In some examples, a Mtb polypeptide or immunogenic fragmentthereof or a polynucleotide encoding the polypeptide or fragment thereofis administered to the subject at doses of about 0.1 μg to 10 mg ofimmunogenic Mtb polypeptide or polynucleotide encoding the polypeptide.Dosages from 0.1 mg to about 100 mg per subject can be used,particularly if the agent is administered to a secluded site and notinto the circulatory or lymph system, such as into a body cavity or intoa lumen of an organ. However, the particular dose can be determined by askilled clinician. The disclosed Mtb polypeptide (or immunogenicfragment thereof) or polynucleotide encoding the polypeptide or fragmentthereof can be administered in one or several doses, for examplecontinuously, daily, weekly, or monthly. When administered sequentially,the time separating the administration of the disclosed Mtb polypeptide(or immunogenic fragment thereof) or a polynucleotide encoding thepolypeptide or fragment thereof can be seconds, minutes, hours, days, oreven weeks.

The mode of administration can be any used in the art. The amount ofagent administered to the subject can be determined by a clinician, andmay depend on the particular subject treated. Specific exemplary amountsare provided herein (but the disclosure is not limited to such doses).

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

We claim:
 1. A method for producing an immune response in a subject,comprising administering to the subject a therapeutically effectiveamount of a viral expression vector comprising a promoter operablylinked to a polynucleotide encoding a polypeptide, wherein thepolypeptide comprises the amino acid sequence set forth as SEQ ID NO:16, thereby inducing the immune response to the polypeptide.
 2. Themethod of claim 1, wherein the polypeptide consists of the amino acidsequence set forth as SEQ ID NO:
 16. 3. The method of claim 1, furthercomprising administering a therapeutically effective amount of anadjuvant to the subject.
 4. The method of claim 1, wherein the subjectis at risk for infection with Mycobacterium tuberculosis (Mtb).
 5. Themethod of claim 1, wherein the viral expression vector is a poxvirusvector.
 6. The method of claim 5, wherein the poxvirus vector is avaccinia virus vector.
 7. The method of claim 6, wherein the vacciniavirus vector is a non-replicating vaccinia virus.
 8. The method of claim5, wherein the poxvirus vector is a modified vaccinia Ankara virus. 9.The method of claim 1, wherein the subject is infected withMycobacterium tuberculosis.
 10. The method of claim 1, wherein thesubject has a latent infection with Mycobacterium tuberculosis.
 11. Themethod of claim 1, wherein the promoter is the thymidine kinasepromoter.
 12. The method of claim 1, wherein the viral expression vectorencodes a carrier protein.
 13. The method of claim 1, wherein the viralexpression vector encodes a cytokine or a co-stimulatory molecule. 14.The method of claim 1, wherein the subject is immunocompromised.
 15. Themethod of claim 1, wherein the subject has primary tuberculosis.